Method for detecting microorganisms

ABSTRACT

Described herein are methods of detecting a wound infection and for detecting the presence or absence of microorganisms, for example, wound pathogens in a sample, by contacting a sample with an enzyme produced and/or secreted by the bacteria, and detecting modification or the absence of modification of the substrate, as an indicator of the presence or absence of the enzyme in the sample. The present invention also features a biosensor for detecting the presence or absence of bacteria in a sample.

REFERENCE TO A “SEQUENCE LISTING”

The sequence listing submitted via EFS, in compliance with 37 CFR§1.52(e)(5), is incorporated herein by reference. The sequence listingtext file submitted via EFS contains the file “98282-000120_ST25.txt”,created on Jul. 23, 2013, which is 3 KB in size.

BACKGROUND OF THE INVENTION

Infection of wounds is a major source of health care expenditure in theUnited States. Approximately 5% of all surgical wounds become infectedwith microorganisms, and that figure is considerably higher (10-20%) forpatients undergoing abdominal surgery. Bacterial species, such asStaphylococci are the most frequently isolated organisms from infectedwounds. This is probably because humans are the natural reservoir forstaphylococci in the environment, with up to 50% of the populationcolonized at any given time. Colonization rates are significantly higherin the hospital setting, both among healthcare workers, and amongpatients. Moreover, the colonizing organisms in the hospital environmentare likely to be resistant to many forms of antimicrobial therapy, dueto the strong selective pressure that exists in the nosocomialenvironment, where antibiotics are frequently used. Staphylococci areusually carried as harmless commensals, however given a breach in theepidermis, they can cause severe, even life threatening infection.

Staphylococci are the most common etiologic agents in surgical woundinfections; others include, but are not limited to Streptococcuspyogenes (S. pyogenes), Pseudomonas aeruginosa (p. aeruginosa),Enterococcus faecalis (E. faecalis), Proteus mirabilis (P. mirabilis),Serratia marcescens (S. marcescens), Enterobacter clocae (E. clocae),Acetinobacter anitratus (A. anitratus), Klebsiella pneumoniae (K.pneumonia), and Escherichia coli (E. coli).

Post-surgical infection due to any of the above organisms is asignificant concern of hospitals. The most common way of preventing suchinfection is to administer prophylactic antibiotic drugs. Whilegenerally effective, this strategy has the unintended effect of breedingresistant strains of bacteria. The routine use of prophylacticantibiotics should be discouraged for the very reason that it encouragesthe growth of resistant strains.

Rather than using routine prophylaxis, a better approach is to practicegood wound management, i.e., keep the area free from bacteria before,during, and after surgery, and carefully monitor the site for infectionduring healing. Normal monitoring methods include close observation ofthe wound site for slow healing, signs of inflammation and pus, as wellas measuring the patient's temperature for signs of fever.Unfortunately, many symptoms are only evident after the infection isalready established. Furthermore, after a patient is discharged from thehospital they become responsible for monitoring their own healthcare,and the symptoms of infection may not be evident to the unskilledpatient.

A system or biosensor that can detect the early stages of infectionbefore symptoms develop would be advantageous to both patients andhealthcare workers. If a patient can accurately monitor the condition ofa wound after discharge from the hospital, then appropriateantimicrobial therapy can be initiated early enough to prevent a moreserious infection.

SUMMARY OF THE INVENTION

It has been found that molecules, for example, proteins secreted bymicroorganisms, such as bacteria or fungi, expressed on the cell surfaceof microorganisms, or expressed on the surface of a cell infected with avirus or prion can serve as markers for the detection of the presence orabsence of the microorganism in a sample, for example, a wound or bodyfluid. Accordingly, the present invention features a method of detectingthe presence or absence of a microorganism in a sample by detecting thepresence or absence of a molecular marker for the microorganism in thesample. In particular, the molecular markers to be detected includeproteins, such as enzymes that are specific to a species ofmicroorganism.

In one aspect, the invention features a method for detecting thepresence or absence of a microorganism in a sample, comprising the stepsof contacting the sample with a detectably labeled substrate for anenzyme produced and/or secreted by the microorganism, under conditionsthat result in modification of the substrate by the enzyme; anddetecting the modification or the absence of the modification of thesubstrate. Modification of the substrate indicates the presence of themicroorganism in the sample, and the absence of modification of thesubstrate indicates the absence of the microorganism in the sample. Inparticular, the substrate can consist of labeled peptide that is cleavedby a protease enzyme to give a signal that can be detected. Furthermore,this peptide can be designed with a particular sequence of amino acidresidues extending from one end of the original substrate peptide as a“tag” for use in covalently coupling the substrate to a surface.

In another aspect, the present invention features a method fordiagnosing the presence or absence of a wound infection in a subject,comprising the steps of a) contacting a sample obtained from a wound ina subject with a detectably labeled substrate for an enzyme producedand/or secreted by a microorganism, under conditions that result inmodification of the substrate by the enzyme; and b) detecting amodification or the absence of a modification of the substrate.Modification of the substrate indicates the presence of a woundinfection in the subject, and the absence of modification of thesubstrate indicates the absence of an infection in the subject.

In yet another aspect, the present invention features a method fordiagnosing the presence or absence of a wound infection in a subject,comprising the steps of a) contacting a wound in a subject with adetectably labeled substrate for an enzyme produced and/or secreted by amicroorganism, under conditions that result in modification of thesubstrate by the enzyme; and b) detecting a modification or the absenceof a modification of the substrate. Modification of the substrateindicates the presence of a wound infection in the subject, and theabsence of modification of the substrate indicates the absence of aninfection in the subject.

In another aspect, the invention features a biosensor for detecting thepresence or absence of a microorganism, for example, a wound-specificbacteria in a sample, comprising a solid support and a detectablylabeled substrate for an enzyme produced and/or secreted by themicroorganism, wherein the substrate is attached to the solid support.

In still another aspect, the present invention features a kit fordetecting a wound infection, comprising a biosensor for detecting thepresence or absence of a microorganism in a sample, and one or morereagents for detecting the presence of the microorganism that is thecausative agent of the wound infection. For example, the reagent can beused to detect an enzyme secreted by the microorganism. In particular,the reagent can be used to detect the modification of the substrate ofthe biosensor.

In yet another aspect, the present invention features a polypeptidecomprising or consisting of the sequence of SEQ ID NO: 1, 2, 3, 4, 5, 9,or 10. These polypeptides are useful for the identification and/ordetection of the presence of wound-specific enzymes as described hereinin a sample. In one embodiment, the polypeptide is detectably labeled.

In another aspect, the present invention features a nucleic acidcomprising or consisting of the sequence of SEQ ID NO: 6, 7, or 8. Thisnucleic acid sequences are useful for the identification and/ordetection of the presence of wound-specific enzymes as described hereinin a sample. In one embodiment, the nucleic acid is detectably labeled.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of the cleavage of a target polypeptide substrate(relative fluorescence) over time (minutes) in samples containing anactive bacterial culture or a water control, substrate, and reactionbuffer. (staph=Staphylococcus aureus; listeria=Listeria monocytogenes;pseudo=Pseudomonas aeruginosa; entero=Enterococcus faecalis;strep=Streptococcus salivarius; Seratia=Serratia marcescens; e.coli=Escherichia coli).

FIG. 2 is a graph of the cleavage of a target polypeptide substrate(relative fluorescence) over time (minutes) in samples containingbuffer, substrate solution only (dye), supernatant (containing nocells), S. marcescens cells, or S. marcescens bacterial culture(mixture).

FIG. 3 is a graph of the cleavage of a target polypeptide substrate(relative fluorescence) over time (minutes) in S. marcescens culturesgrown for 4 hours and diluted to an OD of 1 (log phase); S. marcescenscultures grown for 24 hours and diluted to an OD of 1 (stationaryphase); S. marcescens cultures grown for 6 hours and diluted to an OD of2 (log phase); S. marcescens cultures grown for 24 hours and diluted toan OD of 2 (stationary phase), or control samples containing eitherbuffer or dye.

FIG. 4 is a graph of the cleavage of a target polypeptide substrate(relative fluorescence) in S. marcescens cultures having a pH level of6, 6.4, 6.8, 7.2, 7.6, or 8.0 over time (minutes).

FIG. 5 is a graph of the cleavage of a target polypeptide substrate(relative fluorescence) in samples containing Serratia culture, Serratiaculture plus EDTA, or buffer or substrate solution (dye) only (controls)over time (minutes).

FIG. 6A is a scanned image of the fluorescence of a biosensor containingCell Debris Remover in which the Serratia marcescens specific proteasetarget peptide was not bound to it, in the presence of Serratiaextracts.

FIG. 6B is a scanned image of the fluorescence intensity of a biosensorcontaining Cell Debris Remover in which the Serratia marcescens specificprotease target polypeptide was bound to it, in the presence of Serratiaextracts.

FIG. 6C is a schematic representation of a biosensor for detection of aSerratia marcescens specific protease.

FIG. 7A is a graph of the cleavage (absorbance) of target substrate insamples containing bacterial supernatant, assay substrate (p-nitrophenylcaprate), and reaction buffer at 415 nm overtime (minutes).(SA=Staphylococcus aureus; SE=Staphylococcus epidermidis; SM=Serratiamarcescens; SS=Streptococcus salivarius; EC=Escherichia coli;PA=Pseudomonas aeruginosa; EF=Enterococcus faecalis).

FIG. 7B is a graph of the cleavage of target substrate (Δ Abs) insamples containing S. aureus bacterial supernatant, assay substrate(p-nitrophenyl caprate), and reaction buffer at 415 nm over time(minutes). The reaction buffer consisted of 20 mM Tris (pH 8.3) with 1mM ZnSO₄ added, plus either nothing additional (control), 20% methanol(MeOH), 20% DMSO (DMSO), or 10 mM Triton X-100 (Triton).

FIG. 8 is a graph of the change in absorbance (Δ Abs) of samplescontaining bacterial supernatant, assay substrate solution(p-nitrophenyl-N-acetyl-β-D-glucosaminide), and reaction buffer at 405nm over time (hours). (SA=Staphylococcus aureus; SE=Staphylococcusepidermidis; SM=Serratia marcescens; SS=Streptococcus salivarius;EC=Escherichia coli; PA=Pseudomonas aeruginosa; EF=Enterococcusfaecalis).

FIG. 9 is a graph of the absorbance (Abs 420 nm) of samples containingbacterial supernatant, assay substrate solution(ortho-nitrophenyl-N-acetyl-β-D-galactopyranoside), and reaction bufferat 420 nm over time (hours). (SA=Staphylococcus aureus;SE=Staphylococcus epidermidis; SM=Serratia marcescens; SS=Streptococcussalivarius; EC=Escherichia coli; PA=Pseudomonas aeruginosa;EF=Enterococcus faecalis).

FIG. 10 is a scanned image of a 2.5 cm glass microfiber filter (WhatmanGF/A), soaked with p-nitrophenyl caprate (in ispropanol) to which 4different samples have been applied. In Quadrant #1, Staphylococcusaureus was applied; in Quadrant #2, Staphylococcus epidermidis wasapplied; in Quadrant #3 Streptococcus salivarius was applied, and inQuadrant #4, media was applied as a control. The presence of a yellowdye (gray shading), indicating modification of the substrate by anenzyme in the bacteria is shown in Quadrant #2.

FIG. 11 is a graph of detection of enzymatic cleavage (relativefluorescent intensity) of papa1 (papa), pala1 (pala), and paga1 (paga)over time (seconds) in samples containing Pseudomonas strains P1, PA14(PA), and ZK45 (ZK), or buffer alone, or buffer plus substrate.

FIG. 12 is a graph of detection of enzymatic cleavage (fluorescence) ofpapa1 (papa) over time (seconds) in samples containing buffer only,buffer plus papa1, S. pyogenes plus papa1 (Streptococcus), P. aeruginosastrain PA14 plus papa1 (Pseudomonas), E. coli plus papa1 (E. coli), S.aureus plus papa1 (Staph aureus), S. epidermidis plus papa1 (Staphepidermidis), S. marcescens plus papa1 (Serretia), or E. faecalis pluspapa1 (Enterococcus).

FIG. 13A is a graph of the hydrolysis of labeled propionate substrate(change in absorbance) over time (seconds) for samples containing bufferonly, substrate plus buffer, substrate plus supernatant from P.aeruginosa PA14 grown in tryptic soy media (Prop(TS)); substrate plussupernatant from P. aeruginosa PA14 grown in Brain Heart Infusion media(Prop(BHI)); substrate plus P. aeruginosa PA14 grown in tryptic soymedia (Prop(TS cells)); or substrate plus P. aeruginosa PA14 grown inBrain Heart Infusion media (Prop(BHI cells)).

FIG. 13B is a graph of the hydrolysis of labeled butyrate substrate(change in absorbance) over time (seconds) for samples containing bufferonly, substrate plus buffer, substrate plus supernatant from P.aeruginosa PA14 grown in tryptic soy media (Butyra(TS)); substrate plussupernatant from P. aeruginosa PA14 grown in Brain Heart Infusion media(Butyra(BHI)); substrate plus P. aeruginosa PA14 grown in tryptic soymedia (Butyra(TS cells)); or substrate plus P. aeruginosa PA14 grown inBrain Heart Infusion media (Butyra(BHI cells)).

FIG. 13C is a graph of the hydrolysis of labeled caproate substrate(change in absorbance) over time (seconds) for samples containing bufferonly, substrate plus buffer, substrate plus supernatant from P.aeruginosa PA14 grown in tryptic soy media (Caproa(TS)); substrate plussupernatant from P. aeruginosa PA14 grown in Brain Heart Infusion media(Caproa(BHI)); substrate plus P. aeruginosa PA14 grown in tryptic soymedia (Caproa(TS cells)); or substrate plus P. aeruginosa PA14 grown inBrain Heart Infusion media (Caproa(BHI cells)).

FIG. 14 is a graph of the hydrolysis of labeled propionate estersubstrate (absorbance) over time (seconds) in samples containing buffer,buffer plus substrate (PNP-Propionate) or substrate plus the followingbacteria: PA14 P. aeruginosa strain (Pseudomonas), Serratia, S. aureus(Staph aureus), S. epidermidis (Staph epidermidis), Streptococcus(Streptococcus ), Enterococcus (Enterococcus), E. coli, and S. Pyogenes(Strep P ▴).

FIG. 15 is a scanned image of an agarose gel electrophoresed with 1 Kbladder, DNA control (circular pUC 19 plasmid), or linearized pUC 19 DNAfurther cleaved by DNA metabolism enzymes in Staphylococcus aureus (SA),Enterococcus faecalis (EF), E. coli (EC), Pseudomonas aeruginosa (PA),Streptococcus salivarius (SS), Serratia marcescens (SM), orStaphylococcus epidermidis (SE) for 1 hour, 3 hours, or overnight (0/N).Lane numbers and corresponding samples are indicated.

FIG. 16 is a graph of the DNA metabolic activity (relative fluorescence)of labeled probe over time (seconds) in samples containing buffer, probeonly (DBAF12), or probe plus Enterococcus, S. Salivarius (Strepsalivarius), probe administered BamHI enzyme (BamHI), S. pyogenes (Streppyogenes), Pseudomonas, E. coli, S. aureus (Staph aureus), S.epidermidis (Staph epidermidis) or Serratia.

FIG. 17 A is a graph of cleavage of protease substrate papa2 or papa2C(relative fluorescence) over time (seconds) in samples containingbuffer, buffer plus papa2, buffer plus papa2C, or supernatants fromPseudomonas, E. coli, S. aureus (Staph aureus), S. epidermidis (Staphepidermidis), S. Salivarius (Strep salivarius), S. pyogenes (Streppyogenes), Enterococcus, or Serratia plus papa2.

FIG. 17B is a scanned image of the detection of S. epidermidis and lackof detection of S. pyogenes on a biosensor in which the peptidesubstrates 5-bromo-4-chloro-3indolyl butyrate and5-bromo-4-chloro-3indolyl caprylate were bound to a solid substrate(glass microfiber filter) through hydrophobic interactions.

FIG. 17C is a scanned image of the detection of Pseudomonas (right) on abiosensor in which the peptide substrate papa2 is bound to a solidsubstrate (positively charged membrane) through electrostaticinteractions and exposed to Pseudomonas (right) or BHI media containingno bacteria (left).

FIG. 18 is a graph of the cleavage of peptide substrate papa1 (relativefluorescence) over time in samples containing buffer, buffer plus papa1,control (no bacteria) plus papa1, or porcine wound extracts from pigsinfected with 10³, 10⁴ or 10⁵ P. aeruginosa (pseudomonas) bacteria pluspapa1.

DETAILED DESCRIPTION OF THE INVENTION

As part of their normal growth processes, many microorganisms secrete anumber of enzymes into their growth environment. These enzymes havenumerous functions including, but not limited to, the release ofnutrients, protection against host defenses, cell envelope synthesis (inbacteria) and/or maintenance, and others as yet undetermined. Manymicroorganisms also produce enzymes on their cell surface that areexposed to (and interact with) the extracellular environment. Many ofthese enzymes are specific to the microorganism that secretes them, andas such, can serve as specific markers for the presence of thosemicroorganisms. A system that can detect the presence of these enzymesthat are produced and/or secreted can equally serve to indicate thepresence of the producing/secreting microorganism. Alternatively, asystem that can detect the absence of these enzymes that are producedand/or secreted can equally serve to indicate the absence of theproducing/secreting microorganism. Such a detection system is useful fordetecting or diagnosing an infection, for example, a wound infection.

A microorganism detection test system, as described herein can betailored to detect one specific microorganism by identifying a proteinsuch as a secreted enzyme specific to the microorganism to be detected.Alternatively, a test system can be designed to simultaneously identifymore than one microorganism species (for example, at least 2, at least5, or at least 10 different microorganism species), such as those thatcommonly infect wounds. Identifying those enzymes that are common tocertain classes of pathogenic microorganisms, but which are not presentin non-pathogenic microorganisms is one way to achieve this goal. Suchenzymes can be identified, for example, with a computer basedbioinformatics screen of the microbial genomic databases. By usingenzymes as the basis for detection systems, sensitive tests can bedesigned, since even a very small amount of enzyme can catalyze theturnover of a substantial amount of substrate.

The present invention pertains to the identification of bacterialproteins that are specific for microorganisms that are the causativeagent of a wound, i.e., wound-specific. The proteins can be grouped intoclasses insofar as they represent targets for developing agents fordetecting the bacteria that produce them and present them on the cellsurface or that secrete them. As described herein, proteins were groupedinto nine classes. The presence of a pathogenic bacterium can bedetected by designing a synthetic substrate that will specifically reactwith an enzyme that is present on the surface of the cell or secreted.These synthetic substrates can be labeled with a detectable label suchthat under conditions wherein their respective enzymes specificallyreact with them, they undergo a modification, for example, a visiblecolor change that is observed.

The enzymes that are used in the bacteria detection method of thepresent invention are preferably wound-specific enzymes. As used herein,a wound-specific enzyme is an enzyme produced and/or secreted by apathogenic bacteria, but is not produced and/or secreted by anon-pathogenic bacteria. Examples of pathogenic bacteria include, butare not limited to staphylococcus (for example, Staphylococcus aureus,Staphylococcus epidermidis, or Staphylococcus saprophyticus),streptococcus (for example, Streptococcus pyogenes, Streptococcuspneumoniae, or Streptococcus agalactiae), enterococcus (for example,Enterococcus faecalis, or Enterococcus faecium), corynebacteria species(for example, Corynebacterium diptheriae), bacillus (for example,Bacillus anthracis), listeria (for example, Listeria monocytogenes),Clostlidium species (for example, Clostridium perfringens, Clostridiumtetanus, Clostridium botulinum, Clostridium difficile), Neisseriaspecies (for example, Neisseria meningitidis, or Neisseria gonorrhoeae),E. coli, Shigella species, Salmonella species, Yersinia species (forexample, Yersinia pestis, Yersinia pseudotuberculosis, or Yersiniaenterocolitica), Vibrio cholerae, Campylobacter species (for example,Campylobacter jejuni or Campylobacter fetus), Helicobacter pylori,pseudomonas (for example, Pseudomonas aeruginosa or Pseudomonas mallei),Haemophilus influenzae, Bordetella pertussis, Mycoplasma pneumoniae,Ureaplasma urealyticum, Legionelia pneumophila, Treponema pallidum,Leptospira interrogans, Borrelia burgdorferi, mycobacteria (for example,Mycobacterium tuberculosis), Mycobacterium leprae, Actinomyces species,Nocardia species, chlamydia (for example, Chlamydia psittaci, Chlamydiatrachomatis, or Chlalltydia pneumoniae), Rickettsia (for example,Rickettsia ricketsii, Rickettsia prowazekii or Rickettsia akari),brucella (for example, Brucella abortus, Brucella melitensis, orBrucella suis), Proteus mirabilis, Serratia marcescens, Enterobacterclocae, Acetinobacter anitratus, Klebsiella pneumoniae and Francisellatularensis. Preferably, the wound-specific bacteria is staphylococcus,streptococcus, enterococcus, bacillus, Clostridium species, E. coli,Yersinia, pseudomonas, Proteus mirabilis, Serratia marcescens,Enterobacter clocae, Acetinobacter anitratus, Klebsiella pneumoniae orMycobacterium leprae. For example, the wound-specific enzyme can beproduced and/or secreted by Staphylococcus aureus, Staphylococcusepidermidis, Streptococcus pyogenes, Pseudomonas aeruginosa,Enterococcus faecalis, Proteus mirabilis, Serratia marcescens,Enterobacter clocae, Acetinobacter anitratus, Klebsiella pneumoniaeand/or Escherichia coli.

The wound-specific enzyme may be a lysin (an enzyme that functions tolyse host cells); a cell wall enzyme (an enzyme involved in thesynthesis and turnover of bacterial cell wall components, includingpeptidoglycan), a protease (an enzyme that specifically ornon-specifically cleaves a peptide, polypeptide, or protein), ahydrolase (an enzyme that breaks down polymeric molecules into theirsubunits), a metabolic enzyme (an enzyme designed to perform varioushousekeeping functions of the cell, such as breaking down nutrients intocomponents that are useful to the cell), or a virulence enzyme (anenzyme that is required by the bacterial cell to cause an infection).

Preferably, the enzyme is one or more of the following (the GenBankAccession Number and/or name of an example of each protein is providedin parentheses): autolysin (At1), FemB protein (femB), fmhA protein(fmhA), TcaB protein (tcaB), enterotoxin P (sep), exotoxin 6 (set6),exotoxin 7 (set7), exotoxin 8 (set8), exotoxin 9 (set9), exotoxin 10(set10), exotoxin 11 (set11), exotoxin 12 (set12), exotoxin 13 (set13),exotoxin 14 (set14), exotoxin 15 (set15), Clumping factor B (clfB),BIt-like protein (SA1269), FmhC protein (fmhC(eprh)), enterotoxin SEM(sem), enterotoxin SeN (sen), enterotoxin SeO (seo), leukotoxin LukE(lukE), truncated integrase (SA0356), enterotoxin typeC3 (sec3),enterotoxin Yent1 (yent1), enterotoxin YENT2 (yent2), glycerol esterhydrolase (geh), immunodominant antigen A (isaA), serine protease Sp1B(sp1B), serine protease Sp1C (sp1C), ABC transporter permease (vraG),phosphomevalonate kinase (mvaK2), gamma-hemolysin component B (h1gB),gamma-hemolysin component C (h1gC), tagatose-6-phosphate kinase (lacC),cysteine protease precursor (sspB), 6-phospho-beta-galactosidase (lacG),extracellular enterotoxin L (sel), triacylglycerol lipase precursor(lip), Staphopain, Cysteine Proteinase (SA1725), tagatose1,6-diphosphate aldolase (lacD), gamma-hemolysin chain II precursor(h1gA), enterotoxin homolog (SA1429), mannitol-1-phosphate5-dehydrogenase (mt1D), staphylococcal accessory regulator A (sarA),lactose phosphotransferase system repressor (lacR), capsularpolysaccharide biosynthesis (SA2457), capA, galactose-6-phosphateisomerase LacA subunit (lacA), fibrinogen-binding protein A, clumpingfactor (c1fA), extracellular enterotoxin type G precursor (seg),extracellular enterotoxin type I precursor (sei), leukotoxin, LukD[pathogenicity island SaPIn3] (lukD), fibronectin-binding proteinhomolog (fhb), fibronectin-binding protein homolog (fnbB), holin homolog[Bacteriophage phiN315] (SA1760), similar to D-xylulose reductase(SA2191), secretory antigen precursor SsaA homolog (ssaA), factoressential for expression of methicillin resistance (femA), similar toexotoxin 2 (SA0357), similar to exotoxin 1 (SA1009), similar to exotoxin4 (SA1010), similar to exotoxin 3 (SA1011), staphylococcal accessoryregulator A homolog (sarH3), similar to transaldolase (SA1599), similarto 5-nucleotidase (SA0022),undecaprenyl-PP-MurNAc-pentapeptide-UDPGlcNAc GlcNAc transferase (murG),similar to exonuclease SbcD (SA1180), similar to membrane protein(SA2148), Ser-Asp rich fibrinogen-binding, bone sialoprotein-bindingprotein (sdrC), Ser-Asp rich fibrinogen-binding, bonesialoprotein-binding protein (sdrD), Ser-Asp rich fibrinogen-binding,bone sialoprotein-binding protein (sdrE), similar to oligoendopeptidase(SA1216), similar to MHC class II analog (SA2006), similar totranscription factor (SA0858), probable beta-Iactamase [Pathogenicityisland SaPIn3] (SA1633), similar to NA(+)/H(+) exchanger (SA2228),similar to xylitol dehydrogenase (SA0242), similar to cell wall enzymeEbsB (SA1266), similar to transposase for IS232 (SAS069), similar totransposase for IS232 (SAS070), similar to transport protein SgaT(SA0318), similar to transcription regulator (SA0187), similar to ribosetransporter RbsU (SA0260), similar to regulatory protein PfoR (SA0298),similar to enterotoxin A precursor (SA1430), similar to regulatoryprotein pfoR (SA2320), transposase homolog for IS232 [Pathogenicityisland SaPIn3] (tnp), similar to formate transporter NirC(SA0293),similar to D-octopine dehydrogenase (SA2095), similar to rbs operonrepressor RbsR(SA0261), similar to cell surface protein Map-w (SA0841),similar to fibrinogen-binding protein (SA1OOO), similar tofibrinogen-binding protein (SA1003), similar to fibrinogen-bindingprotein (SA1004), similar to staphylocoagulase precursor (SA0743),similar to ferrichrome ABC transporter (SA0980), similar to peptidebinding protein OppA (SA0849), similar to proton antiporter efflux pump(SA0263), similar to kdp operon sensor protein (kdpD(SCCmec)), similarto secretory antigen precursor SsaA (SA0270), similar to outer membraneprotein precursor (SA0295), similar to deoxyribodipyrimidine photolyase(SA0646), similar to secretory antigen precursor SsaA (SA2097), similarto integral membrane efflux protein (SA2233), similar to secretoryantigen precursor SsaA (SA2332), similar to secretory antigen precursorSsaA (SA2353), similar to transmembrane efflux pump protein (SA0099),similar to multi-drug resistance efflux pump (SA0115), probablespecificity determinant HsdS [Pathogenicity island SaPIn3] (SA1625),similar to ABC transporter ATP-binding protein (SA0339), similar tocobalamin synthesis related protein (SA0642), similar to transcriptionregulator MarR family (SA2060), similar to N-CarbamoylsarcosineAmidohydrolase (SA2438), similar to teichoic acid biosynthesis protein B(SA0243), similar to teichoic acid biosynthesis protein B (SA0247),similar to transcription regulator, RpiR family (SA2108), similar to twocomponent sensor histidine kinase (SA2180), similar tosuccinyl-diaminopimelate desuccinylase (SA1814), similar toextracellular matrix and plasma binding (SA0745), similar totranscription antiterminator BglG family (SA1961), similar to cobalaminsynthesis related protein CobW (SA2368), similar to DNA polymerase II,alpha chain PolC type (SA1710), similar to spermine/spermidineacetyltransferase bIt (SA1931), similar to trimethylamine dehydrogenase(EC 1.5.99.7) (SA0311), similar to AraC/XylS family transcriptionalregulator (SA0622), similar to PTS fructose-specific enzyme IIBCcomponent (SA0320), similar to beta-Iactamase [Pathogenicity islandSaPIn1] (SA1818), similar to 4-diphosphocytidyl-2C-methyl-D-erythritolsynthase (SA0241), similar to synergohymenotropic toxinprecursor—Staphylococcus intermedius (SA1812), similar to bacteriophageterminase small subunit [Pathogenicity island SaPIn1] (SA1820), similarto poly(glycerol-phosphate) alpha-glucosyltransferase (teichoic acidbiosynthesis) (SA0523). The above referenced GenBank Accession Numbersare those corresponding to Staphylococcus aureus proteins. The GenBankAccession Numbers for these proteins from other species are available toone skilled in the art. Such GenBank Accession Numbers can be obtained,for example, by searching the GenBank protein database by the desiredprotein and species name. Alternatively, the Staphylococcus aureusprotein sequence can be obtained using the provided GenBank Accessionnumber and/or protein name, and this sequence can be searched forproteins from other species with similar sequence identity or homology,for example, using the BLAST program described herein. Protein sequencesfrom other species can then be obtained from the search results.

Substrates for use in the present invention include any molecule, eithersynthetic or naturally-occurring that can interact with an enzyme of thepresent invention. Examples of substrates include those substratesdescribed herein, as well as substrates for these enzymes that are knownin the art. Other examples of substrates include Alt derived fluorescentpeptides, for example, PGTKLYTVPW-pyrene (SEQ ID NO: 1) (which can bindto the surface of Staphylococcus; it is predicted that an increase influorescence upon binding would occur); fluorescent peptidoglycans, forexample, flourescent-N-acetylglucosamine-[b-1,4-N acetylmuramic acid,fluorescent-N-acetylmuramyl-L-alanine, or fluorescent-lipoteichoic acid(peptidoglycans over-Iabeled with fluorescein would be quenched fromfluorescing, but following hydrolysis by a wound pathogen wouldfluoresce); and a lipid vesicle containing dye for the detection ofhemolysin (many hemolysins form ordered protein complexes that are poreforming toxins, and can be detected by the release of dye from a lipidvesicle followed by diffusion of the dye onto a hydrophobic solidsubstrate). Such substrates described herein can be obtained fromcommercial sources, e.g., Sigma (St. Louis, Mo.), or can be produced,e.g., isolated or purified, or synthesized using methods known to thoseof skill in the art.

The enzymes of the present invention can modify substrates, for example,proteins or polypeptides by cleavage, and such modification can bedetected to determine the presence or absence of a pathogen in a sample.One method for detecting modification of a substrate by an enzyme is tolabel the substrate with two different dyes, where one serves to quenchthe fluorescence of the other dye by fluorescence energy transfer (FRET)when the molecules, for example, dyes or colorimetric substances are inclose proximity, and is measured by detecting changes in fluorescence.

FRET is the process of a distance-dependent excited state interaction inwhich the emission of one fluorescent molecule is coupled to theexcitation of another. A typical acceptor and donor pair for resonanceenergy transfer consists of 4-[[-(dimethylamino) phenyl]azo]benzoic acid(DABCYL, Dabcyl) and 5-[(2-aminoethylamino]naphthalene sulfonic acid(EDANS, Edans). EDANS is excited by illumination with 336 nm light, andemits a photon with wavelength 490 nm. If a DABCYL moiety is locatedwithin 20 angstroms of the EDANS, this photon will be efficientlyabsorbed. DABCYL and EDANS will be attached to opposite ends of apeptide substrate. If the substrate is intact, FRET will be veryefficient. If the peptide has been cleaved by an enzyme, the two dyeswill no longer be in close proximity and FRET will be inefficient. Thecleavage reaction can be followed by observing either a decrease in thefluorescence of the acceptor or an increases in fluorescence of thedonor. An increase in fluorescence of EDANS can be measured at, forexample, 485 nm or 538 nm.

If the substrate to be modified is a protein, peptide, or polypeptide,the substrate can be produced using standard recombinant proteintechniques (see for example, Ausubel et al., “Current Protocols inMolecular Biology,” John Wiley & Sons, (1998), the entire teachings ofwhich are incorporated by reference herein). In addition, the enzymes ofthe present invention can also be generated using recombinanttechniques. Through an ample supply of enzyme or its substrate, theexact site of modification can be determined, and a more specificsubstrate of the enzyme can be defined, if so desired. This substratecan also be used to assay for the presence of the pathogenic bacteria.

The substrates are labeled with a detectable label that is used tomonitor interactions between the enzyme and the substrate and detect anysubstrate modifications, for example, cleavage of the substrate or labelresulting from such interactions. Examples of detectable labels includevarious dyes that can be incorporated into substrates, for example,those described herein, spin labels, antigen or epitope tags, haptens,enzyme labels, prosthetic groups, fluorescent materials,chemiluminescent materials, bioluminescent materials, and radioactivematerials. Examples of suitable enzyme labels include horseradishperoxidase, alkaline phosphatase, β-galactosidase, andacetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride and phycoerythrin; an example of a chemiluminescent materialincludes luminol; examples of bioluminescent materials includeluciferase, luciferin, and aequorin, and examples of suitableradioactive material include ¹²⁵1, ¹³¹1, ³⁵S and ³H. Other examples ofdetectable labels include Bodipy, Pyrene, Texas Red, IAEDANS, DansylAziridine, IATR and fluorescein. Succimidyl esters, isothiocyanates, andiodoacetamides of these labels are also commercially available. Whendetectable labels are not employed, enzymatic activity can be determinedby other suitable methods, for example detection of substrate cleavagethrough electrophoretic analysis, or other methods known to one skilledin the art.

One example of a preferred detectable label is a chromogenic dye thatallows monitoring of the hydrolysis of the substrate by the bacterialenzyme. An example of such a dye is para-nitrophenol. When conjugated toa substrate molecule, this dye will remain colorless until the substrateis modified by the secreted enzyme, at which point it turns yellow. Theprogress of the enzyme-substrate interaction can be monitored bymeasuring absorbance at 415 nm in a spectrophotometer. Other dyes thatproduce detectable modification, e.g., a visible color change, are knownto those of skill in the art.

The sample in which the presence or absence of bacteria is detected, ora wound infection is diagnosed, can be, for example, a wound, a bodyfluid, such as blood, urine, sputum, or wound fluid (for example, pusproduced at a wound site). The sample can also be any article thatbacteria may be contained on/in, for example, a wound dressing, acatheter, a urine collection bag, a blood collection bag, a plasmacollection bag, a polymer, a disk, a scope, a filter, a lens, foam,cloth, paper, a suture, swab, test tube, a well of a microplate, contactlens solutions, or a swab from an area of a room or building, forexample, an examination room or operating room of a healthcare facility,a bathroom, a kitchen, or a process or manufacturing facility.

The present invention also features a biosensor for detecting a (one ormore, for example, at least 2, at least 5, at least 10, at least 20, atleast 30, at least 50, at least 75, or at least 100) marker proteinenzyme(s) described herein and for notifying a consumer of the presenceof the marker protein. As used herein, a “biosensor” is a device thatincorporates one or more of the above-described substrates, or othersubstrates described herein, and produces a detectable signal uponsensing the presence or absence of bacteria. A biosensor for use inhealthcare settings or home use to detect infected wounds comprising a(one or more) specific substrate(s) that is coupled to a solid supportthat is proximal to a wound or other body fluid that is being monitoredfor bacterial contamination is provided. Preferably, the substrate iscovalently bound to a label and thus has a detection signal that uponproteolysis of the substrate-label bond indicates the presence of thebacteria.

The biosensor is made by first determining the specific substrate of aspecific enzyme characteristic of the bacteria to be detected. Thedetermined specific substrate is labeled with one or more, andpreferably, a plurality of detectable labels, for example, chromatogenicor fluorescent leaving groups. Most preferably, the labeling groupprovides a latent signal that is activated only when the signal isproteolytically detached from the substrate. Chromatogenic leavinggroups include, for example, para-nitroanalide groups. Should thesubstrate come into contact with an enzyme secreted into a wound orother body fluid by bacteria or presented on the surface of a bacterialcell, the enzyme modifies the substrates in a manner that results indetection of such a modification, for example, a change in absorbance,which can be detected visually as a change in color (for example, on thesolid support, such as a wound dressing), or using spectrophotometrictechniques standard in the art.

The biosensor of the present invention also can comprise one or moresubstrates (for example, at least 2, at least 5, at least 10, at least20, at least 30, at least 50, at least 75, or at least 100 substrates)for produced and/or secreted enzymes of pathogenic bacteria. Thebiosensor is a solid support, for example, a wound dressing (such as abandage, or gauze), any material that needs to be sterile or free ofmicrobial contamination, for example, a polymer, disk, scope, filter,lens, foam, cloth, paper, or sutures, or an article that contains orcollects the sample (such as a urine collection bag, blood or plasmacollection bag, test tube, catheter, swab, or well of a microplate).

Typically, the solid support is made from materials suitable forsterilization if the support directly contacts the wound or sample. Inone embodiment of the present invention, the biosensor can be directlycontacted with the wound. In some instances, a sterile covering or layeris used to prevent contamination of the wound or body fluid upon suchdirect contact. If such sterile coverings are used, they will haveproperties that make them suitable for sterilization, yet do notinterfere with the enzyme/substrate interaction. Preferably, the portionof the biosensor that comes into contact with the wound is alsononadherent to permit easy removal of the biosensor from the samplesurface. For example, if the biosensor comprises a wound dressing, thedressing contacts the wound for a time sufficient for the enzymesubstrate to react and then the dressing is removed from the woundwithout causing further damage to the wound or surrounding tissue.

Substrates suitably labeled with detectable labels, for example, achromogenic dye, and attached or incorporated into a sensor apparatus,can act as indicators of the presence or absence of pathogenic bacteriathat secrete the aforementioned enzymes. When more than one substrate isutilized, each may be labeled so as to distinguish it from another (forexample, using different detectable labels) and/or each may be localizedin a particular region on the solid support.

Substrates with hydrophobic leaving groups can be non-covalently boundto hydrophobic surfaces. Alternatively hydrophilic or hydrophobicsubstrates can be coupled to surfaces by disulfide or primary amine,carboxyl or hydroxyl groups. Methods for coupling substrates to a solidsupport are known in the art. For example, fluorescent and chromogenicsubstrates can be coupled to solid substrates using non-essentialreactive termini such as free amines, carboxylic acids or SH groups thatdo not affect the reaction with the wound pathogens. Free amines can becoupled to carboxyl groups on the substrate using, for example, a 10fold molar excess of either N-ethyl-N′-β-dimethylaminopropyl)carbodiimide hydrochloride (EDC) orN-cyclohexyl-N′-2-(4′-methyl-morpholinium) ethyl carbodiimide-p-toluenesulphonate (CMC) for 2 hrs at 4° C. in distilled water adjusted to pH4.5 to stimulate the condensation reaction to form a peptide linkage. SHgroups can be reduced with DTI or TCEP and then coupled to a free aminogroup on a surface with N-e-Maleimidocaproic acid (EMCA, Griffith etal., Febs Lett. 134:261-263, 1981).

One example of a substrate for use in the present invention is apolypeptide comprising or consisting of the amino acid sequence of SEQID NO: 1, 2, 3, 4, 5, 9, or 10, or a polypeptide having at least 50%,60%, 70%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 1,2, 3, 4, 5, 9, or 10, as determined using a sequence comparison programand parameters described herein. Such polypeptides are enzymaticallycleaved by wound specific proteases as described herein.

Another example of a substrate for use in the present invention is apolypeptide comprising or consisting of the nucleic acid sequence of SEQID NO: 6, 7, or 8, or a nucleic acid sequence having at least 50%, 60%,70%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6, 7, or8, as determined using a sequence comparison program and parametersdescribed herein. Such polypeptides are enzymatically cleaved by woundspecific proteases as described herein.

The polypeptides of the invention also encompass fragments and sequencevariants of the polypeptides and nucleic acids described above. Variantsinclude a substantially homologous polypeptide encoded by the samegenetic locus in an organism, i.e., an allelic variant, as well as othervariants. Nucleic acid variants also include allelic variants. Variantsalso encompass polypeptides or nucleic acids derived from other geneticloci in an organism, but having substantial homology to a polypeptide ofSEQ ID NO: 1, 2, 3, 4, 5, 9, or 10 or a nucleic acid of SEQ ID NO: 6, 7,or 8. Variants also include polypeptides or nucleic acids substantiallyhomologous or identical to these polypeptides or nucleic acids butderived from another organism, i.e., an ortholog. Variants also includepolypeptides or nucleic acids that are substantially homologous oridentical to these polypeptides or nucleic acids that are produced bychemical synthesis. Variants also include polypeptides or nucleic acidsthat are substantially homologous or identical to these polypeptides ornucleic acids that are produced by recombinant methods.

The percent identity of two amino acid sequences or two nucleic acidsequences can be determined by aligning the sequences for optimalcomparison purposes (e.g., gaps can be introduced in the sequence of afirst sequence). The amino acids at corresponding positions are thencompared, and the percent identity between the two sequences is afunction of the number of identical positions shared by the sequences(i.e., % identity=# of identical positions/total # of positions×100). Incertain embodiments, the length of the amino acid sequence aligned forcomparison purposes is at least 30%, preferably, at least 40%, morepreferably, at least 60%, and even more preferably, at least 70%, 80%,90%, or 100% of the length of the reference sequence. The actualcomparison of the two sequences can be accomplished by well-knownmethods, for example, using a mathematical algorithm. A preferred,non-limiting example of such a mathematical algorithm is described inKarlin et al., Proc. Natl. Acad. Sci. USA, 90:5873-5877, 1993). Such analgorithm is incorporated into the BLAST programs (version 2.2) asdescribed in Schaffer et aI. (Nucleic Acids Res., 29:2994-3005, 2001).When utilizing BLAST and Gapped BLAST programs, the default parametersof the respective programs can be used. In one embodiment, the databasesearched is a non-redundant (NR) database, and parameters for sequencecomparison can be set at: no filters; Expect value of 10; Word Size of3; the Matrix is BLOSUM62; and Gap Costs have an Existence of 11 and anExtension of 1.

In another embodiment, the percent identity between two amino acidsequences or two nucleic acid sequences can be accomplished using theGAP program in the GCG software package (Accelrys Inc., San Diego,Calif.) using either a Blossom 63 matrix or a PAM250 matrix, and a gapweight of 12, 10, 8, 6, or 4 and a length weight of 2, 3, or 4. In yetanother embodiment, the percent identity between two nucleic acidsequences can be accomplished using the GAP program in the GCG softwarepackage (Accelrys Inc.), using a gap weight of 50 and a length weight of3.

Other preferred sequence comparison methods are described herein.

The invention also encompasses polypeptides having a lower degree ofidentity but having sufficient similarity so as to perform one or moreof the same functions performed by the polypeptide, e.g., the ability toact as a substrate for a Serratia marcescens specific protease.Similarity is determined by conserved amino acid substitution. Suchsubstitutions are those that substitute a given amino acid in apolypeptide by another amino acid of like characteristics. Conservativesubstitutions are likely to be phenotypically silent. Typically seen asconservative substitutions are the replacements, one for another, amongthe aliphatic amino acids Ala, Val, Leu, and Ile; interchange of thehydroxyl residues Ser and Thr; exchange of the acidic residues Asp andGlu; substitution between the amide residues Asn and GIn; exchange ofthe basic residues Lys and Arg; and replacements among the aromaticresidues Phe and Tyr. Guidance concerning which amino acid changes arelikely to be phenotypically silent are found in Bowie et al., Science247: 1306-1310, 1990).

Functional variants can also contain substitution of similar amino acidsthat result in no change or an insignificant change in function.Alternatively, such substitutions may positively or negatively affectfunction to some degree. Non-functional variants typically contain oneor more non-conservative amino acid substitutions, deletions,insertions, inversions, or truncation or a substitution, insertion,inversion, or deletion in a critical residue or critical region, suchcritical regions include the cleavage site for a Serratia marcescensspecific protease.

Amino acids in a polypeptide of the present invention that are essentialfor cleavage by a Serratia marcescens specific protease can beidentified by methods known in the art, such as site-directedmutagenesis or alanine-scanning mutagenesis (Cunningham et al., Science,244: 1081-1085, 1989). The latter procedure introduces a single alaninemutation at each of the residues in the molecule (one mutation permolecule).

The invention also includes polypeptide fragments of the amino acidsequence of SEQ ID NO: 1, 2, 3, 4, 5, 9, or 10 or the nucleic acidsequence of SEQ ID NO: 6, 7, or 8 or functional variants thereof.Fragments can be derived from a polypeptide comprising SEQ ID NO: 1, 2,3, 4, 5, 9, or 10 or a nucleic acid comprising SEQ ID NO: 6, 7, or 8.The present invention also encompasses fragments of the variants of thepolypeptides and nucleic acids described herein. Useful fragmentsinclude those that retain the ability to act as substrates for a woundspecific protease.

Fragments can be discrete (not fused to other amino acids orpolypeptides) or can be within a larger polypeptide. Further, severalfragments can be comprised within a single larger polypeptide. In oneembodiment a fragment designed for expression in a host can haveheterologous pre- and pro-polypeptide regions fused to the aminoterminus of the polypeptide fragment and an additional region fused tothe carboxyl terminus of the fragment.

The biosensors of the present invention can be used in any situationwhere it is desirable to detect the presence or absence of bacteria, andin particular, pathogenic bacteria. For example, bacteria that collectson work surfaces in health care facilities, and in particular inoperating rooms can be detected with a biosensor as described herein. Asubstrate, or more than one substrate, that can be modified by an enzymesecreted by or presented on the surface of a bacteria is labeled andcovalently bound to a collector substrate, such as cotton fibers on thetip of a swab. When more than one substrate is utilized, each may belabeled so as to distinguish it from another (for example, usingdifferent detectable labels) and/or each may be localized in aparticular region on the solid support. The swab tip is used to wipe thesurface suspected of being contaminated by bacteria. The swab tip isplaced in a medium and incubated using conditions that allowmodification of the labeled substrate if an enzyme specific for thebound, labeled substrate(s) is present.

The present invention also features a kit for detecting wound-specificbacteria as described herein. The kit can comprise a solid support, forexample, having a plurality of wells (e.g., a microtiter plate), towhich a detectably labeled substrate is linked, coupled, or attached. Ameans for providing one or more buffer solutions is provided. A negativecontrol and/or a positive control can also be provided. Suitablecontrols can easily be derived by one of skill in the art. A samplesuspected of being contaminated by a pathogen described herein isprepared using the buffer solution(s). An aliquot of the sample,negative control, and positive control is placed in its own well andallowed to react. Those wells where modification of the substrate, forexample, a color change is observed are determined to contain amicrobial pathogen. Such a kit is particularly useful for detecting awound infection in a subject.

Also encompassed by the present invention is a kit that comprises abiosensor, such as a packaged sterilized wound dressing, and anyadditional reagents necessary to perform the detection assay.

The method and/or biosensor of the present invention can be used todetect the presence or absence of any wound-specific enzyme describedherein. For example, the method and/or biosensors can be used to detectthe presence or absence of lipase enzymes secreted by pathogenicbacteria. It has been discovered that certain bacteria secrete lipasesinto their environment as part of their survival and/or virulencemechanisms. The lipases serve to break down lipids in the growthenvironment in order to release nutrients. Lipases may also play a rolein disarming mammalian host defenses during infection. Syntheticsubstrates for these secreted enzymes can be employed to detect thepresence of those pathogenic bacteria that secrete them. By synthesizinglipids attached to dye moieties, it is possible to create substratesthat will change color as they are hydrolyzed by secreted lipases. Thedye molecule can be one of many commercially available molecules thatare colorless when attached to fatty acids, and change color when thesubstrate is cleaved by lipase. An example of such a dye is Rhodamine-110 (available from Molecular probes, Eugene, Oreg.). This color changereaction forms the basis of a bacterial sensor, which can beincorporated into healthcare products including, but not limited to,wound dressings.

In another example, the method and/or biosensor of the present inventioncan be used to detect the presence or absence of autolytic enzymes.Autolysins are enzymes that degrade peptidoglycan, a component of thebacterial cell envelope. Autolytic enzymes serve to break downpeptidoglycan, be it that of the parent organism, as part of celldivision and turnover functions, or as a means to breakdown cell wallsof competing bacteria. When labeled with paranitrophenol, syntheticpeptidoglycan subunits (such as, but not limited to,N-acetyl-β-d-glucosaminide) serve as indicators that can form the basisof a bacterial sensor.

In another example, the method and/or biosensor of the present inventioncan be used to detect the presence or absence of beta-galactosidase onthe surface of bacteria cells. Most bacterial species expressbeta-galactosidase as a cytoplasmic enzyme involved in the metabolism oflactose as an energy source. Certain species of Streptococcus, however,display the enzyme on the surface of the cell. A labeled syntheticmolecule that acts as a substrate for beta-galactosidase, (including,but not limited to ortho nitrophenyll β-D-galactopyranoside (ONPG) couldthus be used as a means of detecting streptococci in the environment.

A method for developing an assay for detecting a pathogenic bacteriathat produces at least one enzyme that is secreted by the cell orpresent on the surface of the cell and a method for using the assay todetect pathogenic bacteria producing the enzyme(s) now follows:

Step 1) Define an amino acid sequence that uniquely identifies theprokaryotic microorganism of interest. Alternatively a (one or more)amino acid sequence that is unique to a specific group of pathogens, forexample, wound-specific pathogens can be determined.

Select an amino acid sequence, for example, a protein, peptide, orpolypeptide (marker sequence) that uniquely characterizes or marks thepresence of the microorganism or group of microorganisms (for example,wound-specific pathogens) of interest. The selection can be performedutilizing a bioinfomatic approach, for example, as described in detailbelow. One or more amino acid sequences that are unique to a specificprokaryotic microorganism are determined.

Step 2) Obtain sufficient enzyme to determine conditions facilitatingoptimal modification of a substrate by the enzyme.

Isolate the enzyme from the extracellular medium in which the pathogenicbacteria to be assayed is growing, or from the cell membrane of thebacteria, using standard protein purification techniques, described, forexample, in Ausubel (supra).

Alternatively, if the genetic sequence encoding the enzyme or thelocation of the genetic sequence encoding the enzyme are unknown,isolate and clone the genetic sequence encoding the marker amino acid ofStep 1, or, first determine the genetic sequence, and then proceed asbefore.

Step 3) Determine the conditions for growth of the prokaryotic organismand for the production of an enzyme presented on the surface of the cellor secreted by the cell.

Determine medium required for growth of the specific prokaryoticmicroorganism of interest and for expression of its unique active enzymeinto the medium. Also determine whether a second molecule, for example,an enzyme is required to convert the specific enzyme from an inactiveprecursor form to an active form. To determine if the enzyme has beensecreted in an active form, a sample of the bacterial culture isprovided with chosen potential substrates and cleavage of thesesubstrates is determined. This can be done, for example, by combiningthe bacteria that produce the enzyme with the substrate in theappropriate media and incubating at 37° C. with gentle shaking. Atpreset times (0.1, 0.3, 1.0, 3.0, 5.0, 24 and 48 hours) the samples arecentrifuged to spin down the bacteria, and a small aliquot is removedfor an SDS-PAGE gel sample. After completion of the time course, thesamples are run on a 10-15% gradient SDS-PAGE minigel. Then, theproteins are transferred to Immobilon Pseq (Transfer buffer, 10% CAPS,10% methanol pH 11.0, 15 V for 30 minutes) using a Bio-Rad semi-drytransblotting apparatus. Following transfer of the proteins, the blot isstained with Coomassie blue R-250 (0.25% Coomassie Brilliant Blue R-250,50% methanol, 10% acetic acid) and destained (high destain for 5minutes, 50% methanol, 10% acetic acid; low destain until complete, 10%methanol, 10% acetic acid) followed by sequencing from the N-terminal.Alternatively, the samples can be run on a mass spectrometer in order tomap the sites of proteolytic cleavage using, for example, a VoyagerElite Mass spectrometer (Perceptive Biosystems, Albertville, Minn.).

Step 4) Identify any specific substrate(s) of the active enzymeprotease. Examples of potential substrates include proteins, peptides,polypeptides, lipids, and peptidoglycan subunits. Label each substratewith a detectable label, for example, a detectable label describedherein, or any other detectable label known in the art.

Step 5) Increase the specificity of the enzyme-substrate interaction(optional) by determining the active or binding site of the enzyme (forexample, using FRET as described above), then determining the geneticsequence useful for producing the active or binding site, and cloningthe determined genetic sequence to generate a more specific substrate.

Step 6) Provide a biosensor comprising one or more of the detectablylabeled substrates identified above for detection of the protease of thepathogenic bacteria of interest.

The substrate can be attached to solid support, for example, a wounddressing, or an article that holds the enzyme and substrate, forexample, a body fluid collection tube or bag, a microplate well, or atest tube. The solid support, if desired, can provide a plurality ofderivatized binding sites for coupling to the substrate, for example,succimidyl ester labeled primary amine sites on derivatized plates(Xenobind plates, Xenopore Corp., Hawthorne, N.J.).

Optionally, unoccupied reactive sites on the solid support are blockedby coupling bovine serum albumin, or the active domain of p26 thereto.p26 is an alpha-crystallin type protein that is used in this case toreduce non-specific protein aggregation. The ability of the p26 proteinto refold heat denatured citrate synthetase before and after coupling tothe surface of the food packaging is used as a control for determiningp26 activity. Alpha-crystallin type proteins were recombinantly producedusing standard recombinant DNA technologies (see Ausubel, supra).Briefly, the plasmid containing the beta sheet-charged core domain ofp26 is electroporated into electrocompetent BL21 (DE3) cells (Bio-Rad E.coli pulser). The cells are grown up to an OD600 of 0.8, then inducedwith 1 mM IPTG for 4 hours. The cells are spun down, and sonicated inlow buffer (10 mM Tris, pH 8.0, 500 mM NaC1, 50 mM Imidizole) to lyse(Virsonic, Virtis, Gardiner, N.Y.). The lysate is spun down at 13,000×gfor 10 minutes, and the supernatant 0.45 and 0.2 μm filtered. Thisfiltrate is loaded onto a Ni-NTA superose column (Qiagen, Valencia,Calif., cat #30410). High buffer (10 mM Tris pH 8.0, 500 mM NaCl, 250 mMImidizole) is used to elute the protein.

Allow the enzyme(s) to come into contact with the substrate(s), andmonitor the reaction for a modification in the detectably labeledsubstrate, as described herein. Modification of the substrate indicatesthat the enzyme produced/secreted by the bacteria is present in thereaction. In addition, the absence of modification of the substrateindicates that the enzyme is not present in the sample. If the bacteriaor enzyme is from a wound, modification of the substrate indicates thatthe bacteria is present in the wound, and that the wound is infected,while the absence of modification of the substrate indicates that theparticular bacteria is not present in the wound, and that the wound isnot infected with that particular bacteria.

EXAMPLES

The present invention will now be illustrated by the following Examples,which are not intended to be limiting in any way.

Example 1 Identification of Wound-Specific Proteins

The TIGR comprehensive microbial resource multi genomic analysis toolslocated at the following Internet site:http://www.tigr.org/tigr-scripts/CMR2Iselect_genomes.spl?showref=true&reforg=O&cutoff=60&logic=AND&showheader=true, as available on Jun. 18, 2001 wereused to analyze the complete genome sequences of the following commonwound pathogen species: Staphylococcus aureus (S. aureus),Staphylococcus epidermidis, Streptococcus pyogenes, Pseudomonasaeruginosa, Enterococcus faecalis, and Escherichia coli. Specifically,each gene in the S. aureus genome was compared for homologs in each ofthe other above specified genomes. Any S. aureus gene that did not havea homolog (with at least 45% identity at the amino acid level) to all 5pathogens, was discarded. The remaining pool contained only those genesthat are common to these six major wound pathogens. These searches wereconducted using the default settings as parameters.

To identify wound-specific genes, the genomes of 47 othernon-wound-pathogenic bacteria were then used to identify those genesthat are common to the wound pathogens, but not to the other 47non-wound infecting bacteria. This screening resulted in the examinationof 132,313 genes that were compared to the wound pathogen genomes. Aftersubtracting those genes with greater than 45% homology to non-woundpathogens, using the same comparison parameters as above, 131 genes ofknown function were identified as wound specific. Subsequent analysis ofthe 131 genes revealed that they fell roughly into the following ninegroups based upon their predicted function:

1. lysins; enzymes that function to lyse host cells or competingbacterial cells.

2. Putative exotoxins; proteins that are homologous to certain secretedtoxins of the staphylococci. These proteins have enzyme signatures fromblock searches of exotoxins including topoisomerase I, synapsin-like,and aminopeptidase.

3. Cell wall machinery; enzymes involved in the synthesis and turnoverof bacterial cell wall components, including peptidoglycan.

4. Matrix binding proteins; proteins that allow bacteria to bind to theextracellular matrix molecules of the host environment (fibronectin andfibrinogen). These proteins have enzyme signatures from block searchesincluding specific recombinase, adenylate cyclases class-I, andNADH-ubiquinone oxidoreductase.

5. Proteases; enzymes that either specifically or non-specificallydigest other protein molecules.

6. Hydrolases; enzymes that break down polymeric molecules into theirsubunits.

7. Metabolic proteins; a broad class of enzymes designed to performvarious housekeeping functions of the cell, such as breaking downnutrients into components that are useful to the cell.

8. Transcription factors; proteins involved in the control of DNAtranscription.

9. Virulence factors; general class of proteins that are required by thebacterial cell to cause an infection. These proteins have enzymesignatures from block searches of virulence factors, including glycosidehydrolases.

The following wound-specific enzymes were identified according to thesequence comparison methods described above (the GenBank AccessionNumber and/or protein name for an example of each protein is provided inparentheses): autolysin (Atl), FemB protein (femB), fmhA protein (fmhA),TcaB protein (tcaB), enterotoxin P (sep), exotoxin 6 (set6), exotoxin 7(set7), exotoxin 8 (set8), exotoxin 9 (set9), exotoxin 10 (set10),exotoxin 11 (set11), exotoxin 12 (set12), exotoxin 13 (set13), exotoxin14 (set14), exotoxin 15 (set15), Clumping factor B (clfB), BIt-likeprotein (SA1269), FmhC protein (fmhC(eprh)), enterotoxin SEM (sem),enterotoxin SeN (sen), enterotoxin SeO (seo), leukotoxin LukE (lukE),truncated integrase (SA0356), enterotoxin typeC3 (sec3), enterotoxinYent1 (yent1), enterotoxin YENT2 (yent2), glycerol ester hydrolase(geh), immunodominant antigen A (isaA), serine protease SplB (splB),serine protease SplC (splC), ABC transporter permease (vraG),phosphomevalonate kinase (mvaK2), gamma-hemolysin component B (hlgB),gamma-hemolysin component C (hlgC), tagatose-6-phosphate kinase (lacC),cysteine protease precursor (sspB), 6-phospho-beta-galactosidase (lacG),extracellular enterotoxin L (sel), triacylglycerol lipase precursor(lip), Staphopain, Cysteine Proteinase (SA1725), tagatose1,6-diphosphate aldolase (lacD), gamma-hemolysin chain II precursor(hlgA), enterotoxin homolog (SA1429), mannitol-1-phosphate5-dehydrogenase (mtID), staphylococcal accessory regulator A (sarA),lactose phosphotransferase system repressor (lacR), capsularpolysaccharide biosynthesis (SA2457), capA, galactose-6-phosphateisomerase LacA subunit (lacA), fibrinogen-binding protein A, clumpingfactor (clfA), extracellular enterotoxin type G precursor (seg),extracellular enterotoxin type I precursor (sei), leukotoxin, LukD[Pathogenicity island SaPln3] (lukD), fibronectin-binding proteinhomolog (fhb), fibronectin-binding protein homolog (fnbB), holin homolog[Bacteriophage phiN315] (SA1760), similar to D-xylulose reductase(SA2191), secretory antigen precursor SsaA homolog (ssaA), factoressential for expression of methicillin resistance (femA), similar toexotoxin 2 (SA0357), similar to exotoxin 1 (SA1009), similar to exotoxin4 (SA1010), similar to exotoxin 3 (SA1O11), staphylococcal accessoryregulator A homolog (sarH3), similar to transaldolase (SA1599), similarto 5-nucleotidase (SA0022),undecaprenyl-PP-MurNAc-pentapeptide-UDPGlcNAc GlcNAc transferase (murG),similar to exonuclease SbcD (SA1180), similar to membrane protein(SA2148), Ser-Asp rich fibrinogen-binding, bone sialoprotein-bindingprotein (sdrC), Ser-Asp rich fibrinogen-binding, bonesialoprotein-binding protein (sdrD), Ser-Asp rich fibrinogen-binding,bone sialoprotein-binding protein (sdrE), similar to oligoendopeptidase(SA1216), similar to MHC class II analog (SA2006), similar totranscription factor (SA0858), probable beta-lactamase [Pathogenicityisland SaPIn3] (SA1633), similar to NA(+)/H(+) exchanger (SA2228),similar to xylitol dehydrogenase (SA0242), similar to cell wall enzymeEbsB (SA1266), similar to transposase for IS232 (SAS069), similar totransposase for IS232 (SAS070), similar to transport protein SgaT(SA0318), similar to transcription regulator (SA0187), similar to ribosetransporter RbsU (SA0260), similar to regulatory protein PfoR (SA0298),similar to enterotoxin A precursor (SA1430), similar to regulatoryprotein pfoR (SA2320), transposase homolog for IS232 [Pathogenicityisland SaPIn3] (tnp), similar to formate transporter NirC(SA0293),similar to D-octopine dehydrogenase (SA2095), similar to rbs operonrepressor RbsR(SA0261), similar to cell surface protein Map-w (SA0841),similar to fibrinogen-binding protein (SA1000), similar tofibrinogen-binding protein (SA1003), similar to fibrinogen-bindingprotein (SA1004), similar to staphylocoagulase precursor (SA0743),similar to ferrichrome ABC transporter (SA0980), similar to peptidebinding protein OppA (SA0849), similar to proton antiporter efflux pump(SA0263), similar to kdp operon sensor protein (kdpD(SCCmec)), similarto secretory antigen precursor SsaA(SA0270), similar to outer membraneprotein precursor (SA0295), similar to deoxyribodipyrimidine photolyase(SA0646), similar to secretory antigen precursor SsaA (SA2097), similarto integral membrane efflux protein (SA2233), similar to secretoryantigen precursor SsaA(SA2332), similar to secretory antigen precursorSsaA(SA2353), similar to transmembrane efflux pump protein (SA0099),similar to multi-drug resistance efflux pump (SA0115), probablespecificity determinant Hsds [Pathogenicity island SaPIn3] (SA1625),similar to ABC transporter ATP-binding protein (SA0339), similar tocobalamin synthesis related protein (SA0642), similar to transcriptionregulator MarR family (SA2060), similar to N-CarbamoylsarcosineAmidohydrolase (SA2438), similar to teichoic acid biosynthesis protein B(SA0243), similar to teichoic acid biosynthesis protein B (SA0247),similar to transcription regulator, RpiR family (SA2108), similar to twocomponent sensor histidine kinase (SA2180), similar tosuccinyl-diaminopimelate desuccinylase (SA1814), similar toextracellular matrix and plasma binding (SA0745), similar totranscription antiterminator Bg1G family (SA1961), similar to cobalaminsynthesis related protein CobW (SA2368), similar to DNA polymerase III,alpha chain PolC type (SA1710), similar to spermine/spermidineacetyltransferase bIt (SA1931), similar to trimethylamine dehydrogenase(EC 1.5.99.7) (SA0311), similar to AraC/XylS family transcriptionalregulator (SA0622), similar to PTS fructose-specific enzyme IIBCcomponent (SA0320), similar to beta-Iactamase [Pathogenicity islandSaPIn1] (SA1818), similar to 4-diphosphocytidyl-2C-methyl-D-erythritolsynthase (SA0241), similar to synergohymenotropic toxinprecursor—Staphylococcus intermedius (SA1812), similar to bacteriophageterminase small subunit [Pathogenicity island SaPIn1] (SA1820), similarto poly(glycerol-phosphate) alpha-glucosyltransferase (teichoic acidbiosynthesis)(SA0523). Some of the above identified enzymes are proteinsthat have known enzymatic activity, while other proteins have enzymesignatures obtained from block searches. Therefore, it is reasonable tobelieve that proteins containing enzymes signatures are suitable for useas enzymes.

Example 2 Preparation of Bacteria for Detection of the Absence orPresence of Bacteria in a Sample

A culture of each of the following bacterial species was grown tosaturation in Brain Heart Infusion (BHI) broth at 37° C. with vigorousshaking (˜200 rpm), using methods that are standard in the art:Staphylococcus aureus; Staphylococcus epidermidis; Serratia marcescens;Streptococcus salivarius; Escherichia coli; Pseudomonas aeruginosa; andEnterococcus faecalis. After overnight growth (to saturation), a 1 mLsample of each culture was obtained, and the cells were removed from theculture supernatant by centrifugation at 12,000×g for 5 minutes. Theremaining culture supernatants were stored on ice until required (lessthan one hour). The bacteria were assayed for the presence or absence ofspecific enzymes as described below. Alternatively, the bacterial cellsare not separated from the culture supernatant, but rather, the assay iscarried out on a sample containing the cells still in suspension intheir culture broth.

Example 3 Detection of Serratia marcescens Using a Protease Assay

A protease is an enzyme that is responsible for the degradation ofproteins by hydrolysis of peptide bonds. A protease can be eithergeneral or specific in its target sequence, depending on its purpose.Pathogenic bacteria secrete some proteases that are specific in natureand target a select protein or peptide for the purpose of either attackof other cells or as a defense mechanism. The target of a specificprotease is identified by the amino acid sequence of the proteinadjacent to the cleavage site.

A Serratia marcescens specific protease was identified based on ahomology search using the sequence of a known sspB protease found in theStaphylococcus species. This protease has homology to the cysteineprotease precursor (sspB) protein of Staphylococcus aureus. Thecorresponding Serratia protease has not been previously characterized.To test for the presence of the specific protease in a bacterialculture, a short target peptide was designed. This target peptide wasderived from a polypeptide substrate previously shown to be cleaved byS. aureus sspB (Chan and Foster, J. Bacteriology 180:6232-6241, 1998).This peptide was capped by a fluorescent dye molecule on one end and byan associated chromophore molecule on the other end. If the absorptionband of the chromophore has sufficient overlap with the absorbance bandassociated with the fluorescence of the dye molecule, the observedfluorescence will be quenched. This phenomenon is known as fluorescenceresonance energy transfer (FRET) and can be used to determine thedistance between the FRET donor and acceptor molecules. Upon cleavage ofthe peptide the fluorescent indicator is released from the proximity ofthe quencher and the fluorescence increase is measured. Thus, thepresence or absence of a protease that targets the peptide in the samplecan be determined by detecting fluorescence emitted by the cleavedpeptide.

The specificity of the protease for Serratia marcescens was determinedby detecting cleavage of a target polypeptide, using FRET, by theprotease in a number of different bacterial pathogen samples. Thebacterial pathogen samples used in this study were all taken fromovernight cultures grown in brain heart infarction (BHI) media at 37°C., as described herein. The bacterial pathogens chosen for this studywere: Staphylococcus aureus, Listeria monocytogenes, Pseudomonasaeruginosa, Enterococcus faecalis, Streptococcus salivarius, Serratiamarcescens, and Escherichia coli. The target peptide substrate used totest for the Serratia marcescens specific protease was as follows:Dabsyl-NEAIQEDQVQYE-Edans (SEQ ID NO: 2), and was prepared usingstandard methods known to one skilled in the art. A substrate solutioncontaining 1 mg/mL to 5 mg/mL of target peptide substrate in 1:1water/dimethylsulfoxide (DMSO) was prepared. The reaction buffer usedwas 20 mM Tris (pH 7.5) with 200 mM NaCl. The assay was carried outusing 3 L of substrate solution, 7 L of bacterial culture medium, and140 L of reaction buffer, for a total volume of 150 L of assay mixture.The assay mixture was loaded into individual wells of a microtiter plateand the plate was placed in a fluorimeter. The narrow band filters inthe fluorimeter were centered at 305 nm for excitation and 485 nm forfluorescence emission readings. The samples were incubated at 37° C. andthe fluorescence of each sample was measured at time points taken every10 minutes. A sample containing substrate, reaction buffer, and water inplace of the bacterial control was used as a negative control.

The results of this study are shown in FIG. 1, which is a graph showingthe change in relative fluorescence intensity over time for samplescontaining an active bacterial culture (grown overnight in BHI media) ora water control, substrate, and reaction buffer. Culture mediumcontaining S. marcescens cells reacted with the substrate, giving riseto an increase in fluorescence intensity over the course of thereaction. None of the other bacterial samples, including theStaphylococcus aureus sample, were distinguishable from the controlreaction containing water. Furthermore, the reaction took only minutesto distinguish the sample containing S. marcescens from the otherstested here. Thus, this assay can be used as a specific detection systemfor the presence of S. marcescens in a sample.

Example 4 The Serratia Specific Protease is Exported from the Cell

Another experiment was performed to determine whether the Serratiaspecific protease is found on the cell surface or if the cell exportsthe protein into the media. If the protease is exported, it may then beable to diffuse through the media towards its substrate. If not, thenthe bacteria would have to be in contact with the substrate to allowdetection of the presence of the pathogen in the sample. Such an assaywas carried out as described below.

An S. marcescens culture was grown overnight in BHI. The cells wereseparated from the culture media by centrifugation so that the activityassociated with each could be measured. The cell pellets were washed andthen re-dissolved in buffer to give the original volume. The proteolyticactivity of the washed cells was then compared to the proteolyticactivity of the supernatant media in the Serratia protease assay,described above. Seven L aliquots of each sample were run using theassay conditions described above (using 3 L of substrate solution and140 L of 20 mM Tris buffer (pH 7.5) with 200 mM NaCI added).

The results obtained for this experiment are shown in FIG. 2, which is agraph of the relative fluorescence of the target peptide, indicating theamount of peptide cleavage in samples containing buffer, substratesolution only (dye), supernate (containing no cells), S. marcescenscells, or S. marcescens bacterial culture (mixture) over time. Thesample labeled “mixture” contains the original overnight growth mediacontaining active Serratia cultures that was used to generate thesamples of cells and supernatant. As shown in FIG. 2, the proteaseactivity obtained from the supernatant sample was nearly equivalent tothe protease activity for the overnight culture. The sample obtainedfrom the cell pellet did not show any activity and was in the same rangeas the buffer and dye controls. These results show that the Serratiaprotease is exported from the cell and can diffuse away from the cellsurface into the media. These results thus indicate that a Serratia cellin a sample does not have to come into direct contact with its targetsubstrate in order to be detected.

Example 5 Activity of the Serratia Specific Protease Under VariousGrowth Conditions

The stage of enzyme production and/or export from a pathogen is also afactor to be taken into consideration when designing methods andbiosensors for detecting wound specific-pathogens in a sample. Thesynthesis and export of a bacterial protease can be regulated by thegrowth conditions. Some proteases produced by pathogenic bacteria areinduced under growth limiting conditions. To investigate the growthconditions by which the S. marcescens specific protease was expressed,the activity of the protease (cleavage of the target polypeptide)produced by cells that were grown overnight to stationary phase wascompared to the activity of the protease produced by cells that had beenactively growing in log phase conditions for several hours.

The optical density (OD) of the samples used in this experiment wasadjusted by dilution in order to account for cell density differencesduring growth. The overnight cultures were diluted with BHI media togive samples with an OD of 1 or 2 for comparative activity measurements.S. marcescens specific protease activity was assessed as describedherein, by measuring the relative fluorescence of the cleaved targetpolypeptide. The volume of the bacterial cell cultures, the substratesolution (dye), and the reaction buffer added remained constant. Theassay conditions used for this experiment were the same as describedabove. The results of these studies are shown in FIG. 3, which is agraph of the relative fluorescence of cleaved target polypeptide by S.marcescens cultures grown for 4 hours and diluted to an OD of 1 (logphase); S. marcescens cultures grown for 24 hours and diluted to an ODof 1 (stationary phase); S. marcescens cultures grown for 6 hours anddiluted to an OD of 2 (log phase); S. marcescens cultures grown for 24hours and diluted to an OD of 2 (stationary phase), or control samplescontaining either buffer or dye. As shown in FIG. 3, the activityobserved for the log phase samples were both higher than thecorresponding stationary phase samples of the same OD, however theprotease was present and readily detectable in both the log andstationary phases of cell growth. These results demonstrate the abilityof the S. marcescens protease assay to be both rapid and specific. Inaddition, the assay was shown to be robust under various pathogen growthconditions.

Example 6 Activity of the Serratia Specific Protease Under Various pHConditions

The conditions for which a pathogen detection assay is suitable wasstudied in order to determine the range of applicability of the assay.Some of the relevant parameters include pH, temperature, saltconcentration, and nutrient availability. Physiological data is knownfor some of these parameters, however the conditions in a wound may varyin such things as pH and nutrient availability. To address these issues,further experiments were performed to determine the pH range for theSerratia protease activity.

The pH dependence experiment was carried out using the supernatantobtained by centrifugation of an overnight culture of S. marcescens andthe FRET assay described herein. The assay solution was buffered with 20mM sodium phosphate at 6 different pH levels: pH 6, 6.4, 6.8, 7.2, 7.6,and 8.0. The salt concentration used was constant at 200 mM NaCL. Thevolumes of both the supernatant and the substrate solution (dye)remained the same as those used in the studies described above. Theresults of this study are shown in FIG. 4, which is a graph of therelative fluorescence of the samples having different pH levels overtime. The optimum pH for Serratia protease is 6.8, however the activitydoes not vary much over the pH range studied here. The useful range ofthis assay extends from below pH 6 to above pH 8. This indicates thatthe Serratia protease is robust under a wide range of pH conditions, andtherefore appears to be a good assay target for a wound infectionsensor.

Example 7 The Serratia Specific Protease is not a Metalloprotease

Several types of proteases are found in bacteria and are categorized bythe catalytic group used in the active site. The most common bacterialproteases are the serine protease, the cysteine protease, and themetalloprotease. The metalloprotease is so named because it contains acatalytic zinc ion at the active site. The bound zinc ion is generallylabile and can be removed by chelation. Therefore, reduction of theactivity by addition of a chelator to the assay buffer indicates theprotease is most likely a metalloprotease.

To determine whether the Serratia specific protease is ametalloprotease, the following study was performed. One mMethylenediaminetetraacetic acid (EDTA) was added to the standard assaysolution (20 mM Tris (pH 7.5) with 200 mM NaCl) used in theabove-described FRET assay, and its effect on protease activity(measured as a decrease in relative fluorescence activity of theprotease) was determined. FIG. 5 is a graph of the effect of EDTA on theprotease activity, measured by relative fluorescence of the targetpeptide in samples containing Serratia culture, Serratia culture plusEDTA, or buffer or substrate solution (dye) only (controls). Noreduction in activity was found for the sample containing EDTA whencompared to native activity, indicating that the Serratia protease isprobably a serine protease or a cysteine protease.

Example 8 A Biosensor for Detection of Serratia marcescens

An example of a biosensor for the detection of a Serratia marcescens (S.marcescens) specific protease, and therefore, for the detection ofSerratia marcescens now follows. The Serratia marcescens specific targetpeptide substrate was bound to a surface with a weak positive charge,such as fibrous cellulose lightly substituted with DEAE (Cell DebrisRemover, Whatman, Inc.). This matrix was placed under a film, forexample, a clear bandage, as shown in FIG. 6C, and fluorescence in thepresence of Serratia was detected. The efficacy of this biosensor wasdemonstrated by exposing the biosensor containing Cell Debris Remover inwhich the Serratia marcescens specific protease target peptide was notbound to it (FIG. 6A; negative control biosensor), or a biosensorcontaining Cell Debris Remover in which the Serratia marcescens specificprotease did contain the target polypeptide (FIG. 6B) to Serratiamarcescens extract. Very little fluorescence was emitted from thecontrol biosensor, while fluorescence was readily detected in thebiosensor containing the target peptide. These results demonstrate thatsuch a solid phase wound infection biosensor consisting of the peptidebound to Cell Debris Remover can be used to detect the Serratia pathogenin a wound or any other sample or surface containing the pathogen.

The above studies demonstrate the identification of a novel peptidesubstrate that is specific for S. marcescens. The activity associatedwith this protease appears to be novel. The studies described hereinalso indicate that the S. marcescens specific protease is secreted andthe protease is present in all phases of growth. In addition, thedetection assay is robust under various pH conditions, demonstratingthat this S. marcescens specific protease can be used for the fordetection of S. marcescens in a sample.

Example 9 Detection of the Presence of Staphylococcus aureus andStaphylococcus epidermidis Using a Lipase Assay

Certain bacteria secrete lipases into their environment as part of theirsurvival and/or virulence mechanisms. The lipases serve to break downlipids in the growth environment in order to release nutrients. Lipasesmay also play a role in disarming mammalian host defenses duringinfection. Lipases fall into the category of secreted hydrolases fromthe list outlined above.

To test for the presence or absence of lipases secreted by bacteria, thelipase substrate p-nitrophenyl caprate, obtained from Sigma (CatalogNo.: N-0252). This lipase substrate consists of capric acid, aten-carbon lipid molecule, esterified with the dye para nitrophenol(detectable label) as described above. The substrate was dissolved inisopropyl alcohol at a concentration of 8 mM (2.35 mg/mL). A reactionbuffer containing 20 mM Tris (pH 8.5) was also prepared.

To perform the lipase assay, 80 L of reaction buffer, 10 L of culturesupernatant from each bacteria species described in Example 2, and 10 Lof assay substrate were added to a well of a 96-well microplate. Eachbacterial species was assayed individually, and the assays wereperformed in triplicate. The 96-well plate was incubated at 37° C. for60 minutes. At 5 minute intervals during the incubation period,absorbance at 415 nm indicating modification of the enzyme by a lipasesecreted by the cells, was automatically measured using a BioRadBenchmark Microplate reader.

FIG. 7A is a graph showing the results of the lipase assay, measured asthe absorbance at 415 nm over a period of 60 minutes. As shown in FIG. 7A, labeled substrate incubated with culture supernatants fromStaphylococcus epidermidis (S. epidermidis) and Staphylococcus aureus(S. aureus) showed a dramatic color change, as detected by absorbance at415 nm after just a few minutes. Other bacterial samples showed no colorchange, although a slight increase in turbidity contributed to theabsorbance at 415 nm. This lipase assay is therefore suitable for thedetection of Staphylococcus aureus or Staphylococcus epidermidis in asample. Modification of p-nitrophenyl caprate by a bacterial sampleindicates that the bacteria can be Staphylococcus. In addition, theabsence of modification of p-nitrophenyl caprate by a bacterial samplecan indicate that the bacteria is not Staphylococcus.

Other major wound pathogens also secrete lipolytic enzymes into theirgrowth medium (Rosenau and Jaeger, Biochime, 82:1023, 2000), so it mayseem surprising that other organisms do not react with this substrate.However, it is known that bacterial lipases do show strong substratespecificity with regard to the chain length of the fatty acid theyhydrolyze (Van Kampen et al., BBA, 1544:229, 2001) and to the locationin a lipid layer. The degree that the environment of the substrateaffects the efficiency of hydrolysis depends on the particular enzyme.To test this for the lipase from S. aureus, the above-described lipaseassay was performed in the presence of several detergent and solventadditives. The reaction buffer consisted of 20 mM Tris (pH 8.3) with 1mM ZnSO₄ added, plus either nothing additional (control), 20% methanol,20% DMSO, or 10 mM Triton X-100. As shown in FIG. 7B, the hydrolysisrate was found to increase in the presence of organic solvents such asmethanol and DMSO, and in the presence of the detergent Triton X-100.

Example 10 Detection of the Presence of Enterococcus faecalis Using anAutolysin Assay

Autolysins are enzymes that degrade peptidoglycan, a component of thebacterial cell envelope. Autolytic enzymes serve to break downpeptidoglycan, be it that of the parent organism, as part of celldivision and turnover functions, or as a means to breakdown cell wallsof competing bacteria. Autolysins fall into the category of “cell wallmachinery” in the list of categories detailed above.

To test for the presence of autolysins in bacterial cell culturesupernatants, the synthetic autolysin substratep-nitrophenyl-N-acetyl-b-D-glucosaminide (PNP-AGA), a substratecontaining a dye that is detected at 415 nm when the substrate ismodified, was dissolved in 50% DMSO to a final concentration of 20 mM toform a substrate solution. Modification of this substrate can bedetected by measuring the change in absorbance at 405 nm. A reactionbuffer containing 20 mM NaPO₄ and 200 mM NaCI (pH 7.0) was alsoprepared. The assay was performed as follows. Five-hundred L of reactionbuffer, 50 L of substrate solution (20 mM PNP-AGA), and 450 L of testsample (bacterial supernatant as described in Example 2, or water(control)) were added to a reaction tube. Each bacterial species wasassayed individually. The samples were incubated at 20° C. for 7 hours.The progress of modification of the labeled substrate was monitored byabsorbance at 405 nm.

FIG. 8 is a graph showing the change in absorbance of samples containingbacteria supernatant (or water), substrate and reaction buffer over time(in hours). Supernatant from Enterococcus faecalis (E. faecalis)cultures reacted with the substrate, giving rise to a color change overthe course of the reaction. None of the other culture supernatants weredistinguishable from the water control. Thus, this assay can be used asa specific detection system for Enterococcus faecalis. Modification ofPNP-AGA by a bacterial sample indicates that the bacteria can beEnterococcus. In addition, the absence of modification of PNP-AGA by abacterial sample can indicate that the bacteria is not Enterococcus.

Example 11 Detection of the Presence of Streptococcus Salivarius Using aBeta-Galactosidase Assay

Most bacterial species express beta-galactosidase as a cytoplasmicenzyme for the metabolism of lactose as an energy source. Certainspecies of Streptococcus, however, display the enzyme on the surface ofthe cell. A labeled synthetic molecule that acts as a substrate forbeta-galactosidase, (for example, ortho nitrophenylb-D-galactopyranoside (ONP-GP)) could thus be used as a means ofdetecting streptococci in the environment.

To determine the presence or absence of bacteria in a sample, mid-logphase bacterial supernatants were obtained as described in Example 2. Asubstrate solution containing the labeled synthetic substrateortho-nitrophenyl-b-D-galactopyranoside (ONPG) dissolved in 50% DMSO toa concentration of 20 mM was prepared. In addition, a reaction solutioncontaining 20 mM NaPO₄ (pH 7) with 200 mM NaCl. The beta-galactosidaseassay was carried out as follows. Five hundred μL of reaction buffer,450 L of bacterial supernatant, and 50 μL of substrate solution werecombined in a reaction tube to give 1 mL total volume. A controlcontained 450 μL of water in place of the supernatant. The samples wereincubated at 37° C. and absorbance at 420 nm was measured hourly.

FIG. 9 is a graph of the absorbance of the samples at 420 nm over time(in hours). As shown in FIG. 9, most culture supernatants did not showsigns of beta-galactosidase activity. Streptococcus salivarius (S.salivarius) supernatant however, did react with the substrate, mostlikely because of surface expressed beta-galactosidase. Since most ofthe enzyme remains attached to the cell surface, the reactivity may bedue to enzyme that has cleaved from the surface, or from cells thatcarried over into the supernatant. Accordingly, alternatively, the assaycan be performed on a cell suspension, rather than on the cellsupernatant.

In this assay, Streptococcus salivarius reacted with the substrate,while other species did not. This assay forms the basis of a specifictest for Streptococcus salivarius. Modification of ONPG by a bacterialsample indicates that the bacteria can be Streptococcus. In addition,the absence of modification of ONP-GP by a bacterial sample can indicatethat the bacteria is not Streptococcus.

Example 12 A Biosensor for Detection of Staphylococcus epidermidis

An example of a biosensor for the detection of an enzyme secreted byStaphylococcus epidermidis (S. epidermidis), and therefore, for thedetection of Staphylococcus epidermidis now follows. A 100 mM solutionof 20 mM p-nitrophenyl Caprate (in ispropanol) was applied to a 2.5 cmglass microfiber filter (Whatman GF/A). The isopropanol was allowed toevaporate at room temperature for 30 minutes, leaving the substratebound to the filter. After the filter was completely dry, a single dropof bacterial culture was applied to each quadrant of the filter. InQuadrant #1, Staphylococcus aureus was applied; in Quadrant #2,Staphylococcus epidermidis was applied; in Quadrant #3 Streptococcussalivarius was applied, and in Quadrant #4, media was applied as acontrol. The filter was incubated at 37° C. for 30 minutes, anddetection of a yellow dye, indicating modification of the substrate byan enzyme in the bacteria was detected. As shown in FIG. 10, nomodification of the labeled substrate was detected in Quadrants #1, #3,or #4. Modification of the labeled substrate was detected in Quadrant#2. These results demonstrate how a biosensor can be used to detect thepresence or absence of a microorganism in a sample.

Example 13 Detection of the Presence of Pseudomonas aeruginosa Using aProtease Assay or a Lipase Assay

Three peptide substrates of Pseudomonas aeruginosa (P. aeruginosa) wereidentified and synthesized. The three peptides are shown in Table 1.

TABLE 1 Peptide Origin Function papa1 PepA Exoprotein pala1 LasAElastase Gene Cluster paga1 Poly-gly from StaphylococcusPathogen:Pathogen Interactions

The peptide substrates used here were labeled with the fluorescent probeedans (5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid) and thequencher dye molecule dabcyl((4-(4-(dimethylamino)phenyl)azo)benzoicacid). The labeled peptide sequences used were as follows:

(SEQ ID NO: 3) PAPA1 Edans - KAAHKSALKSAE - Dabcyl (SEQ ID NO: 4) PALA1Edans - KHLGGGALGGGAKE - Dabcyl (SEQ ID NO: 5) PAGA1Edans - KHLGGGGGAKE - Dabcyl.

Additional substrates tested for their suitability in detecting P.aeruginosa were the para-nitrophenyl lipid ester substrates shown inTable 2.

TABLE 2 Lipid Ester Origin Length Propionate synthetic C3 Butyratesynthetic C4 Caproate synthetic C6 and others synthetic up to C18

The para-nitrophenyl lipid ester substrates were used at a concentrationof 10 mM dissolved in isopropanol.

Protease Assay

A protease assay for detecting the presence of P. aeruginosa was carriedout as follows. Three strains of P. aeruginosa bacteria (P1, a “studentfriendly” strain, PA14, the accepted standard strain for virulencemodels, and ZK45, a clinical isolate for Children's Hospital in Boston,Mass.) were grown in an incubator overnight at 37° C. in 5 mL of BHI(Brain Heart Infusion) media. The resulting cultures were spun down bycentrifugation and the supernatants were collected. The assays wereperformed in 20 mM tris buffer (pH 7.5) with 150 mM NaCI added. Thereactions were carried out with 3 μL of supernatant and 7 μL of labeledsubstrate (as indicated in FIG. 11) in 100 μL total volume at 37° C. Thereaction was followed by measuring absorbance at 485 nm on afluorimetric plate reader. The results are shown in FIG. 11. As shown inFIG. 11, the papa1 peptide substrate was cleaved by Pseudomonas.

This protease assay was repeated using various strains of bacteria,including S. pyogenes, P. aeruginosa strain PA14, S. epidermidis, S.marcescens, and E. faecalis and the peptide substrate papa1. As shown inFIG. 12, the protease assay was specific for detection of P. aeruginosa.

A lipase assay for detecting the presence of P. aeruginosa in a samplewas carried out as follows. Bacteria from the P. aeruginosa strain PA14were grown in an incubator overnight at 37° C. in 5 mL of TS (TrypticSoy Broth with dextrose) or BHI (Brain Heart Infusion) media. Theresulting cultures were separated into two samples: one sample was usedas a culture of cells and media, and the other sample was spun down bycentrifugation and the supernatant was collected. The lipase assays wererun in 20 mM tris buffer (pH 7.5) with 150 mM NaCI added. The reactionwas carried out with 10 μL of labeled substrate (propionate (FIG. 13A),butyrate (FIG. 13B), or caproate (FIG. 13C)) in 100 μL total volume at37° C. In the indicated samples, the reaction included 10 μL ofbacterial supernatant or 10 μL of bacterial cell culture. The reactionwas followed by measuring absorbance at 415 nm on a colorimetric platereader. The results are shown in FIGS. 13A-13C). As shown in FIGS.13A-13C, nitrophenyl substrates (C3-C6) are suitable for detecting P.aeruginosa.

Additional studies involving detection of P. aeruginosa using the abovedescribed lipase assay were carried out as follows. The PA14 P.aeruginosa strain, Serratia, S. aureus, S. epidermidis, Streptococcus,Enterococcus, E. coli, and S. Pyogenes were each grown in an incubatorovernight at 37° C. in 5 mL of BHI (Brain Heart Infusion) media. Theresulting cultures were spun down by centrifugation and the supernatantwas collected. This set of assays was run in 20 mM tris buffer (pH 7.5)with 150 mM NaCI added. The reaction was carried out with 10 μL ofsubstrate in 100 μL total volume at 37° C. In the indicated samples(FIG. 14) the reaction included 10 μL of bacterial supernatant. Thereaction was followed by measuring the absorbance at 415 nm on acolorimetric plate reader. The results are shown in FIG. 14.

As shown in FIG. 14, over time, P. aeruginosa demonstrated the greatestactivity on the substrate. The reaction conditions can varied to furtherseparate the reactivity of Pseudomonas in comparison to other species,if a faster reaction time is desired. A large change in the reactivityof a lipase enzyme can be achieved, for example, by modification of thereaction solution to more closely match the environment of the lipidmembrane.

Additional studies can be carried out on the substrates to examine thecross reactivity of the substrate with additional reaction agents orwith the types of molecules that could be present in a woundenvironment, for example, serum during the detection of P. auruginosa.If the substrates cross-react with serum, it may be desirable to modifythe substrate or the reaction conditions, using methods known to one ofskill in the art, to lower the cross reactivity.

Example 14 Detection of the Presence of Microorganisms By Detecting DNAMetabolism Enzymes

As described herein, DNA metabolism enzymes is a class of enzymesidentified in the bioinformatics search of genes that are in commonamong wound pathogens. Based on this knowledge, the types of DNAmetabolism activities (exonuclease and endonuclease) that can bedetected with wound pathogens grown in culture were determined asfollows. Ten μg of pUC19 DNA was linearized by digestion with EcoRIenzyme. Ten mL overnight cultures of S. aureus, E. faecalis, E. coli, P.aeruginosa, S. salivarius, S. marcescens, and S. epidermidis were thengrown. Five μL of DNA was added to 70 μL of bacterial cells. The sampleswere then incubated for the time periods of 1 hr, 3 hr, and overnight.At the indicated time intervals an aliquot of the sample was removed andplaced in a new tube. The reaction was stopped with 10× DNA samplebuffer and the samples were stored at −20° C. before running on a 1.2%TBE agarose gel (80V, constant power). The DNA metabolism activities bythe various bacterial cultures are shown in FIG. 15.

As shown in FIG. 15, all bacteria tested had some DNA metabolismactivity. Bacteria with pronounced endonuclease activity included S.aureus, S. epidermidis, E. faecalis, and P. aeruginosa. Bacteria withstrong exonuclease activity include S. marcescens, P. aeruginosa, E.coli, and Streptococcus. In addition, Staphylococcus (aureus orepidermidus) had little exonuclease activity.

Another method for detecting DNA metabolism activity is to generatesynthetic colorimetric and fluorescent DNA probes that can indicate DNAhydrolysis (exonuclease and endonuclease activity). The method wascarried out as follows. Two complementary oligonucleotides weregenerated. One oliogonucleotide was labeled with self-quenchingfluorescent labels, while the other primer remained unlabeled. Thesequences of the oligonucleotides were as follows: Unlabeled Sequence5′-CCTCTCGAGGATCCACTGAATTCCT-3 (SEQ ID NO: 6); and Labeled SequenceFL-5′-AGGAATTCAGTGGATCCTCGAGAGG-3′-FL (SEQ ID NO: 7). Bacteria (E.faecalis. S. Salivarius, S. pyogenes, P. aeruginosa, E. coli, S. aureus,S. epidermidis, and S. marcescens) were grown in an incubator overnightat 37° C. in 5 mL of BHI (Brain Heart Infusion) media. The culture wasspun down by centrifugation and the supernatant was collected. Thefluorescent labeled primer and its unlabeled complement were dissolvedin water at a concentration of approximately 1 mg/mL. The primers wereheated to melting temperature for 2 minutes at 92° C., then allowed toanneal for 5 minutes at 43° C. After annealing, the DNA substrate wasadded to the reaction buffer (20 mM tris (pH 7.4) with 150 mM NaCI) andincubated at 37° C. for 10 minutes. The reaction was carried out with 7μL of culture supernatant and 3 μL of DNA substrate in 100 μL totalvolume at 37° C. The reaction was followed using a fluorimetric platereader using an excitation wavelength of 485 nm and an emissionwavelength of 538 nm. The results of this assay are shown in FIG. 16.The labeled DNA probe detected specific DNA metabolic activityassociated with Enterococcus. Another probe that can be used to detectthe presence of bacteria with DNA metabolism activity is:Rh-5′-AGGAATTCAGTGGATCCTCGAGAGG-3′-FL (SEQ ID NO: 8) and its complement,which, when annealed, is designed to give both colorimetric andfluorimetric signals upon cleavage of the DNA sequence.

Example 15 Development of Biosensor Surfaces

The attachment of molecules to surfaces can be performed by the use ofseveral different types of interactions. Typically, proteins can beattached to surfaces using hydrophobic, electrostatic, or covalentinteractions. There are many commercially available membranes and resinswith a variety of surface properties. Surfaces can also be chemicallymodified to provide the required surface properties.

Commercially available transfer membranes exist for protein and peptidebinding. They consist of positively and negatively charged polymers suchas ion exchange membrane disc filters and resins. Nitrocellulosemembranes offer hydrophobic and electrostatic interactions. Glass fibermembranes offer a hydrophobic surface that can easily be chemicallymodified to add functional groups. There are also modified polymermembranes that offer reactive functional groups that covalently bindproteins and peptides.

It is also possible to utilize various functional groups on membranes orresins and a crosslinking agent to covalently link to proteins.Crosslinking reagents contain two reactive groups, thereby providing ameans of covalently linking two target functional groups. Commonfunctional groups to target on proteins are amine, thiol, carboxylicacid, and alcohol groups that are used to form intramolecularcrosslinks. Crosslinking agents can be homobifunctional orheterobifunctional and a selection of crosslinking agents of variouslengths are commercially available.

Initially the peptides studied were designed as substrates for bacterialassay development using fluorescence energy transfer (Edans and Dabcyl)for detection. Papal, which is selective for Pseudomonas, is an exampleof such a substrate, and is described herein.

In order to develop substrates specifically for surface immobilization,several versions of the papa1 peptide substrate were made. The peptideswere designed to include lysine groups (amine functional group) at oneend of the peptide in the case of papa2. The addition of two lysinegroups (KK) at one end of the peptide serve as a “tag” and providesuitable groups for attachment to surfaces through techniques such aselectrostatic interactions or through covalent attachment. The peptidepapa3 was designed to include a cysteine group (C) and three histidinegroups (HHH) at one end. The addition of a cysteine provides anothersuitable group or tag to perform covalent attachments through the thiolgroup. The inclusion of three histidine groups also provides thepotential for attachment to nickel resins.

The peptide sequences for papa1 was modified as shown:

(SEQ ID NO: 3) papa1 (dabcyl-K)AAHKSALKSA(E-edans) (SEQ ID NO: 9) papa2KKAS(E-edans)AAHKSALKSAE(K-dabcyl) (SEQ ID NO: 10) papa3CHHHAS(E-edans)AAHKSALKSAE(K-dabcyl)

The pre-peptide tags were added to the original papa1 sequence, as shownabove, to allow for attachment to a surface.

The protease assay described herein for detection of P. aentginosa wasrun with the modified version of papa1. Bacteria (Pseudomonas, E. coli,S. aureus, S. epidermidis, S. salivarius, S. pyogenes, Enterococcus, andSerratia) were grown in an incubator overnight at 37° C. in 5 mL of BHI(Brain Heart Infusion) media. Each of the resulting cultures was spundown by centrifugation and the supernatant was collected. The assayswere run in 20 mM tris buffer (PH 7.4) with 150 mM NaCI added. Thereaction was carried out with 7 μL of supernatant and 3 μL of peptidesubstrate (5 mg/mL in water) in 100 μL total volume at 37° C. Thereaction was followed on a fluorimetric plate reader using an excitationwavelength of 355 nm and an emission wavelength of 485 nm. The resultsare shown in FIG. 17A. As shown in FIG. 17 A, this protease assay showedthe greatest fluorescence in the sample containing Pseudmnonas.

Hydrophobic interactions make use of the non-ionic packing that occursin a polar solvent such as water and such interactions can be used inthe production of biosensors for detection of pathogens. The substrates5-bromo-4-chloro-3indolyl butyrate and 5-bromo-4-chloro-3indolylcaprylate can be spotted onto a glass microfiber filter to make asensor. Upon spotting the filter with a small amount of Staphylococcus(aureus or epidermidis) culture medium the color of the filter will turnblue in approximately 15 minutes. An example of this assay is shown inFIG. 17B, where the dark spot corresponding to S. epidermidis indicatesdetection of that pathogen in this assay. The assay did not detect S.pyogenes.

Electrostatic interactions make use of the charges on the peptide orchromophore to bind it to a surface to make a biosensor. For example,ion exchange membranes with a strong negative (ICE450) or positive(SB-6407) charge are available from Pall Gelman Laboratory, Ann Arbor,Mich. It is possible to bind the peptide substrates through interactionswith their charged groups. The peptide substrate papa2, as describedherein, was spotted onto a positively charged membrane and exposed toPseudomonas culture medium. As shown in FIG. 17C, upon cleavage of thepeptide yellow fluorescence (indicated by a bright spot, right side ofFIG. 17C) was observed.

Metal chelate (affinity binding) interactions can provide a strongerbond to biological molecules. A his-tag built into the peptide substratecan be used to allow linkage to a nickel binding resin. The resin isincubated with a suitable culture for 30 minutes at 37° C. Aftercentrifugation the buffer is removed and the pelleted resin is imaged.The fluorescence produced by the peptide is then detected.

Lysine peptide tags, for example, papa2 can be used to link to a surfacesuch as UltraBind™ (pall Gelman Laboratory, Ann Arbor, Mich.). UltraBindis a polyethersulfone membrane that is modified with aldehyde groups forcovalent binding of proteins. Proteins are directly reacted with theUltraBind surface. It is also possible to link proteins or peptides tothe surface using cross linker chains. For example, the carbodiimide,EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, hydrochloride) iscommonly used to link carboxylic acid groups to amines. The linking ofthe peptide with a cross linking agent allows the choice of a linkerchain to extend the peptide off the surface of the membrane while stillcovalently binding it. The linking of the peptide through a cross linkercan be optimized to make the peptide available to the bacterial enzymes.This allows for optimization of the reaction time of the biosensor sincepeptide availability is directly related to this parameter.

Example 16 Detection of Pseudomonas in Porcine Wound Fluid

In order to test for the presence of enzymatic activity in a woundbacterial protease assay for detection of P. aeruginosa was performed onsamples obtained from wound infections made in pigs. The bacteria weregrown in an incubator overnight at 37° C. in 5 mL of Brain HeartInfusion (BHI) media. Each of the resulting cultures was diluted intosodium phosphate buffer at pH 7.2 with 150 MM NaCI (PBS buffer) to givesamples containing 10⁵, 10⁴ and 10³ bacteria total in 100 μL.Immediately after surgery to create a series of partial thickness woundswas performed on the pigs, the wound surfaces were treated with calciumchloride solution for a short period and then patted dry. A buffersolution containing diluted bacterial cultures was placed on the surfaceof the wounds. The wounds were then covered and the infections wereallowed to grow for a period of 3 days. After the dressing was removedfrom the pigs and the wounds were scored for degree of inflammation, 100μL of PBS buffer was added to the wound surface and the extracted woundfluid was recovered by pipette. Each of the samples was split in halfand 50 μL were used to inoculate BHI plates and the other 50 μL wereplaced in plastic tubes and immediately frozen at −80° C.

The buffer used to dilute the wound fluid in the protease assays wasPBS.

The reaction was performed in a 96-well microtiter plate. Thefluorimetric assays for P. aeruginosa was carried out with 20 L offreshly thawed bacterial culture and 5 μL of papa1 peptide substrate (5mg/mL in water) diluted into buffer to give 100 μL total volume at 37°C. The reaction was followed on a fluorimetric plate reader using anexcitation wavelength of 355 nm and an emission wavelength of 528 nm.The reaction was followed for 1 hour and the results are shown in FIG.18.

As shown in FIG. 18, the reactivity of the P. aeruginosa protease wasretained in the wound fluid under these reaction conditions. The samplescontaining P. aeruginosa were detectable using this assay.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A method for detecting the presence or absence of a pathogenicbacterium in a sample of wound fluid obtained from a subject, comprisingthe steps of: a) contacting the sample with a detectably labeledsubstrate for an enzyme produced and/or secreted by each ofStaphylococcus aureus, Staphylococcus epidermidis, Streptococcuspyogenes, Pseudomonas aeruginosa, Enterococcus faecalis and Escherichiacoli, wherein said enzyme is selected from the group consisting of alysin, an exotoxin, a cell wall enzyme, a matrix binding enzyme, aprotease, a hydrolase, a virulence factor enzyme, and a metabolicenzyme, wherein said enzyme is not produced and/or secreted by anonwound-pathogenic bacterium under conditions that result inmodification of said substrate by said enzyme; and b) detecting themodification or the absence of the modification of said substrate,wherein modification of said substrate indicates the presence of saidpathogenic bacterium in said sample, and wherein the absence ofmodification of said substrate indicates the absence of said pathogenicbacterium in said sample.
 2. The method of claim 1, wherein saidpathogenic bacterium is Serratia marcescens.
 3. The method of claim 1,wherein said pathogenic bacterium is Enterococcus faecalis.
 4. Themethod of claim 1, wherein said hydrolase is a lipase.
 5. The method ofclaim 1, wherein said lysin is an autolysin.
 6. The method of claim 1,wherein said metabolic enzyme is beta-galactosidase.
 7. A method fordetecting the presence or absence of a wound infection in a subject,comprising the steps of: a) contacting a sample of wound fluid from asubject with a detectably labeled substrate for an enzyme producedand/or secreted by each of Staphylococcus aureus, Staphylococcusepidermidis, Streptococcus pyogenes, Pseudomonas aeruginosa,Enterococcus faecalis and Escherichia coli, wherein said enzyme isselected from the group consisting of a lysin, an exotoxin, a cell wallenzyme, a matrix binding enzyme, a protease, a hydrolase, a virulencefactor enzyme, and a metabolic enzyme, wherein said enzyme is notproduced and/or secreted by a non-wound-pathogenic bacterium, underconditions that result in modification of said substrate by said enzyme;and b) detecting the modification or the absence of the modification ofsaid substrate, wherein modification of said substrate indicates thepresence of a wound infection in said subject, and wherein the absenceof modification of said substrate indicates the absence of a woundinfection in said subject.
 8. The method of claim 7, wherein saidhydrolase is a lipase.
 9. The method of claim 7, wherein said lysin isan autolysin.
 10. The method of claim 7, wherein said metabolic enzymeis beta-galactosidase.
 11. A biosensor for detecting the presence orabsence of a microorganism in a sample, said biosensor comprising asolid support and a detectably labeled substrate specific for an enzymeproduced and/or secreted by said microorganism, said substrate attachedto said solid support.
 12. The biosensor of claim 11, wherein the solidsupport comprises a material required to be free of microbialcontaminants.
 13. The biosensor of claim 11, wherein said solid supportis a selected from the group consisting of a wound dressing, a containerfor holding body fluids, a disk, a scope, a filter, a lens, foam, cloth,paper, a suture, and a swab.
 14. The biosensor of claim 13, wherein saidcontainer for holding body fluids is selected from the group consistingof a urine collection bag, a blood collection bag, a plasma collectionbag, a test tube, a catheter, and a well of a microplate.
 15. Thebiosensor of claim 11, wherein said microorganism is a wound-specificbacterium.
 16. The biosensor of claim 15, wherein said bacterium isselected from the group consisting of Staphylococcus aureus,Staphylococcus epidermidis, Streptococcus pyogenes, Pseudomonasaeruginosa, Enterococcus faecalis, Proteus mirabilis, Serratiamarcescens, Enterobacter cloacae, Acetinobacter anitratus, Klebsiellapneumonia, and Escherichia coli.
 17. The biosensor of claim 15, whereinsaid wound-specific bacterium is Serratia marcescens.
 18. The biosensorof claim 15, wherein said wound-specific bacterium is Pseudomonasaeruginosa.
 19. The biosensor of claim 15, wherein said wound-specificbacterium is Staphylococcus aureus.
 20. The biosensor of claim 15,wherein said wound-specific bacterium is Enterococcus faecalis.
 21. Thebiosensor of claim 15, wherein said wound-specific bacterium isStaphylococcus epidermidis.
 22. The biosensor of claim 11, wherein saidbiosensor directly contacts said wound.
 23. A kit for detecting a woundinfection, comprising a biosensor for detecting the presence or absenceof a microorganism in a sample, said biosensor comprising a solidsupport and a detectably labeled substrate specific for an enzymeproduced and/or secreted by said microorganism, wherein said substrateis attached to said solid support, and one or more reagents fordetecting the enzyme produced and/or secreted by a microorganism causingsaid wound infection.